Immunologically significant herpes simplex virus antigens and methods for using same

ABSTRACT

The invention provides HSV antigens that are useful for the prevention and treatment of HSV infection. Disclosed herein are epitopes confirmed to be recognized by T-cells derived from herpetic lesions. T-cells having specificity for antigens of the invention have demonstrated cytotoxic activity against cells loaded with virally-encoded peptide epitopes, and in many cases, against cells infected with HSV. The identification of immunogenic antigens responsible for T-cell specificity provides improved anti-viral therapeutic and prophylactic strategies. Compositions containing antigens or polynucleotides encoding antigens of the invention provide effectively targeted vaccines for prevention and treatment of HSV infection.

[0001] This application claims the benefit of U.S. provisional patentapplications Nos. 60/308,923, filed Jul. 31, 2001, and 60/309,428, filedAug. 1, 2001, the entire contents of each of which are incorporatedherein by reference.

[0002] This application is related to U.S. patent application Ser. No.09/672,595, filed Sep. 28, 2000, and U.S. patent application Ser. No.09/368,770, filed August 5, 1999, and to U.S. provisional patentapplications Nos. 60/095,724, filed Aug. 7, 1998, 60/157,181, filed Sep.30, 1999, No. 60/203,660, filed May 12, 2000, and No. 60/218,104, filedJul. 13, 2000, the entire contents of each of which are incorporatedherein by reference. Throughout this application various publicationsare referenced. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to describe more fully the state of the art to which thisinvention pertains.

TECHNICAL FIELD OF THE INVENTION

[0003] The invention relates to molecules, compositions and methods thatcan be used for the treatment and prevention of HSV infection. Moreparticularly, the invention identifies epitopes of HSV proteins that canbe used for methods, molecules and compositions having the antigenicspecificity of HSV-specific T cells, and in particular, of CD8+ as wellas CD4+ T cells.

BACKGROUND OF THE INVENTION

[0004] Cellular immune responses are required to limit the severity ofrecurrent HSV infection in humans. Initial genital HSV-2 infections areprolonged and severe, while recurrences are less severe and morefrequently asymptomatic. Resolution of primary HSV-2 infection isassociated with infiltration of antigen-specific T cells, including CD8+cytotoxic T lymphocytes (CTLs). Serial lesion biopsy studies ofrecurrent HSV-2 infection in humans has shown a shift to CD8+predominance as lesions mature and correlation of local CTL activitywith virus clearance (Koelle, D M et al., J. Clin. Invest. 1998,101:1500-1508; Cunningham, Ala. et al., J. Clin. Invest. 1985,75:226-233). Thus, HSV antigens recognized by CD8+ CTL can be used fornovel therapies and vaccines.

[0005] The complete DNA sequence of herpes simplex virus (HSV) isapproximately 150 kb and encodes about 85 known genes, each of whichencodes a protein in the range of 50-1000 amino acids in length. Unknownare the immunogenic epitopes within these proteins, each epitopeapproximately 9-12 amino acids in length, that are capable of elicitingan effective T cell immune response to viral infection.

[0006] There remains a need to identify specific epitopes capable ofeliciting an effective immune response to HSV infection. Suchinformation can lead to the identification of more effective immunogenicantigens useful for the prevention and treatment of HSV infection.

SUMMARY OF THE INVENTION

[0007] The invention provides HSV antigens, polypeptides comprising HSVantigens, polynucleotides encoding the polypeptides, vectors, andrecombinant viruses containing the polynucleotides, antigen-presentingcells (APCs) presenting the polypeptides, immune cells directed againstHSV, and pharmaceutical compositions. The pharmaceutical compositionscan be used both prophylactically and therapeutically. The antigens ofthe invention are recognized by T cells recovered from herpetic lesions.The invention additionally provides methods, including methods forpreventing and treating HSV infection, for killing HSV-infected cells,for inhibiting viral replication, for enhancing secretion of antiviraland/or immunomodulatory lymphokines, and for enhancing production ofHSV-specific antibody. For preventing and treating HSV infection, forenhancing secretion of antiviral and/or immunomodulatory lymphokines,for enhancing production of HSV-specific antibody, and generally forstimulating and/or augmenting HSV-specific immunity, the methodcomprises administering to a subject a polypeptide, polynucleotide,recombinant virus, APC, immune cell or composition of the invention. Themethods for killing HSV-infected cells and for inhibiting viralreplication comprise contacting an HSV-infected cell with an immune cellof the invention. The immune cell of the invention is one that has beenstimulated by an antigen of the invention or by an APC that presents anantigen of the invention. A method for producing such immune cells isalso provided by the invention. The method comprises contacting animmune cell with an APC, preferably a dendritic cell, that has beenmodified to present an antigen of the invention. In a preferredembodiment, the immune cell is a T cell such as a CD4+ or CD8+ T cell.

[0008] In one embodiment, the invention provides a compositioncomprising an HSV polypeptide. In one embodiment, the polypeptidecomprises a U_(L)49 protein or a fragment thereof. In a preferredembodiment, the fragment of a U_(L)49 protein comprises amino acids14-22, 21-35, 45-59, 49-57, 49-63, 105-190, 177-220 or 193-208 ofU_(L)49 or a substitutional variant thereof. In another embodiment, thepolypeptide comprises an ICP0 protein or a fragment thereof In oneembodiment, the fragment of an ICP0 protein comprises amino acids92-101, 92-105, 288-307 or 743-751 of ICP0 or a substitutional variantthereof. In another embodiment, the polypeptide comprises a U_(L)48protein or a fragment thereof. In one embodiment, the fragment of aU_(L)48 protein comprises amino acids 185-197, 209-221, 288-307 or430-449 of VP16 (U_(L)48) or a substitutional variant thereof.

[0009] Also provided is an isolated polynucleotide that encodes apolypeptide of the invention, and a composition comprising thepolynucleotide. The invention additionally provides a recombinant virusgenetically modified to express a polynucleotide of the invention, and acomposition comprising the recombinant virus. In preferred embodiments,the virus is a vaccinia virus, canary pox virus, HSV, lentivirus,retrovirus or adenovirus. A composition of the invention can be apharmaceutical composition. The composition can optionally comprise apharmaceutically acceptable carrier and/or an adjuvant.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1A is a schematic representing organization of the HSV genomein the region of 0.67-0.73 map units. Boundaries are approximate.HSV-1×HSV-2 intertypic recombinant viruses (IRV) are also shown. HSV-2DNA is indicated by a solid line; HSV-1 DNA by a dashed line, andindeterminate regions by a multiple line. The HSV-2 BamH I w fragmentused for expression cloning is also shown.

[0011]FIG. 1B is a bar graph showing proliferative responses of T-cellclones (TCC) to the indicated IRV. Data are delta CPM [³H] thymidineincorporation compared to media alone, which was less than 500 cpm ineach case.

[0012]FIG. 2 is an immunoblot showing determination of the HSV viralphenotype of the U_(L)49 gene product (VP22) of IRV DX32. Lysates ofmock-infected cells and cells infected with the viral strains DX32,HSV-1 or HSV-2 were separated by SDS-PAGE, blotted, and probed withVP22-specific mAb. The molecular weights (kD) of marker proteins areshown at right.

[0013]FIG. 3A is a bar graph showing T-cell proliferation elicited byvarious peptide epitopes in VP22 of HSV-2 using TCC 4.2E1.Antigen-presenting cells (APC) were autologous EBV-LCL. Antigensincluded β-galactosidase and fusion proteins used at 10 μg/ml andpeptides used at 3 μM. Data are delta cpm [³H] thymidine incorporationcompared to media alone, which was less than 500 cpm in each case.

[0014]FIG. 3B is a bar graph showing T-cell proliferation elicited byvarious peptide epitopes in VP22 of HSV-2 using TCC 1.L3D5.10.8. APCwere autologous PBMC. Antigens included β-galactosidase and fusionproteins used at 10 μg/ml and peptides used at 1 μM. Data are delta cpm[³H] thymidine incorporation compared to media alone, which was lessthan 500 cpm in each case.

[0015]FIG. 3C is a bar graph showing T-cell proliferation elicited byvarious peptide epitopes in VP22 of HSV-2 using TCC ESL4.9. APC wereautologous PBMC. Antigens included β-galactosidase and fusion proteinsused at 10 μg/ml and peptides used at 1 μM. Data are delta cpm [³H]thymidine incorporation compared to media alone, which was less than 500cpm in each case.

[0016]FIG. 4 shows secretion of IFN-γ by lesion-derived CD8⁺ T cellclones in response to COS-7 cells transfected with HLA class I heavychain cDNA and infection with HSV-2 strain 333. Values are mean ofduplicate IFN-γ secretion into medium, measured by ELISA.

[0017]FIG. 5 shows secretion of IFN-γ by lesion-derived CD8⁺ T cellclone 5491.2000.48 in response to COS-7 cells co-transfected with HLAB*0702 cDNA and the indicated HSV-2 DNA fragments. All HSV-2 genes arefull length except for U_(L)47, which is was tested in two segmentsencoding the indicated amino acids. Values are mean of duplicate IFN-γsecretion into the medium as measured by ELISA.

[0018]FIG. 6 shows lysis by lesion-derived CD8 clones of autologous LCLloaded with HSV-2 peptides at the indicated concentrations. Data arepercent specific ⁵¹Cr release at E:T 20:1. , Lysis by clone5101.1999.23 of targets loaded with U_(L)47 551-559;

, clone 1874.1991.22 and U_(L)47 289-298; ▾, clone 1874.1997.51 and ICP092-101; ∇, clone 5491.2000.48 and U_(L)49 49-57. Lysis of mock-loadedtargets was <5% specific release for each clone.

[0019]FIG. 7 shows cytolytic activity of CD8 CTL clones againstcutaneous cells. Top, U_(L)49-specific clone 5491.2000.48, HLAB*0702-expressing fibroblasts from subject SJ, and peptide U_(L)49 aa49-47. Middle, U_(L) 47-specific clone 1874.1991.22, HLA A*0201-bearingfibroblasts from subject 1874, and peptide U_(L) 47 aa 551-559. Left,Lysis of non-IFN-γ-treated fibroblasts infected at the indicated MOI for2 h before assay; right, fibroblasts pretreated for 3 days with 500 U/mlIFN-γ and then infected or treated with peptide. In each case,HLA-mismatched target fibroblasts had <5% specific release at E:T 2:1,6:1, and 20:1. Bottom, HLA A*0201-bearing keratinocytes are used astarget cells. Left, U_(L) 47-specific clone 5101.1999.23 and peptideU_(L)47 aa 289-298; right. U_(L) 47-specific clone 1874.1991.22 andpeptide U_(L) 47 aa 551-559. HLA A*0201-bearing keratinocytes were mocktreated or treated with 500 U/ml IFN-γ and then infected for 2 or 18 hwith HSV-2 at MOI 25 or loaded with peptide for 90 min.

[0020] FIGS. 8A-E show the HLA types of the donors used in Example 5.

[0021]FIG. 9 shows the CD8+ T cell peptide-screening hits.

[0022]FIG. 10 shows the results of peptide screening for donor AD116.

[0023]FIG. 11 shows the results of peptide screening for donors EB5491,TM10062 and HV5101.

[0024]FIG. 12 shows results of peptide screening for donors AD104,AD116, AD120 and D477.

[0025]FIG. 13 is a schematic representation of the positive genomicclone isolated from Sau3A I library of HSV-2 DNA (second line), whichcontained part of the ICP0 gene. The genomic clone was transfected intocells and primer A used for cDNA synthesis. The exon-1/exon2 C-A (fifthline) and HLA B45 cDNAs stimulated interferon-gamma secretion from Tcell clone (TCC) RW51 after transfection into Cos-7 cells. Exon-1 B-CcDNA (fourth line) was negative.

[0026]FIG. 14 is a bar graph showing CTL activity of RW51 againstvaccinia ICP0 and indicated concentrations of synthetic ICP0 92-105.Four-hour ⁵¹Cr release assay with effector:target ratio 10:1.Spontaneous release all <20%.

[0027]FIG. 15 is a graph showing CTL activity of RW51 against indicatedconcentrations of synthetic ICP0 92-101. Four-hour ⁵¹Cr release assaywith effector:target ratio 10:1. Spontaneous release all <20%.

[0028]FIG. 16 is a graph showing CTL activity of lymphocytes subject RW,derived from peripheral blood and stimulated with a peptide of HSV-2ICP0 amino acids 92-101. Four-hour ⁵¹Cr release assay witheffector:target ratio of 10:1. Spontaneous release <20%. For each pairof bars, the upper bar represents data from a lesion-derived CD8 cloneand the lower bar represents data from PBMC stimulated with peptide.

[0029]FIG. 17 shows confirmation of HLA restricting allele, HSV-2reactivity, and IFN-gamma secretion by lesion CD8 clones.

[0030]FIG. 18 shows peptide dose-response for lesion CD8 cloneRW.1997.51 worked up by expression cloning.

[0031]FIG. 19 is a line graph showing HLA restriction element for T-cellclone BM.17 response to peptide 437-449 of VP16 (U_(L)48) of HSV-2.Proliferative responses are plotted versus concentration of viralpeptide. Antigen presenting cells are EBV-LCL that are either autologous(closed circles), homozygous for HIA DQB1*0501 (open triangles), orhomozygous for HLA DQB1*0201 (squares).

DETAILED DESCRIPTION OF THE INVENTION

[0032] The invention provides HSV antigens that are useful for theprevention and treatment of HSV infection. Disclosed herein are antigensand/or their constituent epitopes confirmed to be recognized by T-cellsderived from herpetic lesions. In some embodiments, T-cells havingspecificity for antigens of the invention have demonstrated cytotoxicactivity against virally infected cells. The identification ofimmunogenic antigens responsible for T-cell specificity facilitates thedevelopment of improved anti-viral therapeutic and prophylacticstrategies. Compositions containing antigens or polynucleotides encodingantigens of the invention provide effectively targeted vaccines forprevention and treatment of HSV infection.

[0033] Definitions

[0034] All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

[0035] As used herein, “polypeptide” includes proteins, fragments ofproteins, and peptides, whether isolated from natural sources, producedby recombinant techniques or chemically synthesized. Polypeptides of theinvention typically comprise at least about 6 amino acids, andpreferably at least about 15 amino acids.

[0036] As used herein, “HSV polypeptide” includes HSV-1 and HSV-2,unless otherwise indicated. References to amino acids of HSV proteins orpolypeptides are based on the genomic sequence information regardingHSV-2 as described in A. Dolan et al., 1998, J. Virol. 72(3):2010-2021.As noted below, the predicted polypeptide sequence of ICP0 of HSV-2based on sequencing RNA from cells transfected with a fragment of ICP0differs from the published sequence by the omission of amino acid Q26.

[0037] As used herein, “substitutional variant” refers to a moleculehaving one or more amino acid substitutions or deletions in theindicated amino acid sequence, yet retaining the ability to bespecifically recognized by an immune cell. The amino acid sequence of asubstitutional variant is preferably at least 80% identical to thenative amino acid sequence, or more preferably, at least 90% identicalto the native amino acid sequence. Typically, the substitution is aconservative substitution. One method for determining whether a moleculecan be specifically recognized by an immune cell is the cytotoxicityassay described in D. M. Koelle et al., 1997, Human Immunol. 53:195-205.Other methods for determining whether a molecule can be specificallyrecognized by an immune cell are described in the examples providedhereinbelow, including the ability to stimulate secretion ofinterferon-gamma or the ability to lyse cells presenting the molecule.An immune cell will specifically recognize a molecule when, for example,stimulation with the molecule results in secretion of greaterinterferon-gamma than stimulation with control molecules. For example,the molecule may stimulate greater than 5 pg/ml, or preferably greaterthan 10 pg/ml, interferon-gamma secretion, whereas a control moleculewill stimulate less than 5 pg/ml interferon-gamma.

[0038] As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

[0039] As used herein, “expression control sequence” means a nucleicacid sequence that directs transcription of a nucleic acid. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. The expression control sequenceis operably linked to the nucleic acid sequence to be transcribed.

[0040] The term “nucleic acid” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogs of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally occurring nucleotides.

[0041] As used herein, “antigen-presenting cell” or “APC” means a cellcapable of handling and presenting antigen to a lymphocyte. Examples ofAPCs include, but ate not limited to, macrophages, Langerhans-dendriticcells, follicular dendritic cells, B cells, monocytes, fibroblasts andfibrocytes. Dendritic cells are a preferred type of antigen presentingcell. Dendritic cells are found in many non-lymphoid tissues but canmigrate via the afferent lymph or the blood stream to the T-dependentareas of lymphoid organs. In non-lymphoid organs, dendritic cellsinclude Langerhans cells and interstitial dendritic cells. In the lymphand blood, they include afferent lymph veiled cells and blood dendriticcells, respectively. In lymphoid organs, they include lymphoid dendriticcells and interdigitating cells.

[0042] As used herein, “modified” to present an epitope refers toantigen-presenting cells (APCs) that have been manipulated to present anepitope by natural or recombinant methods. For example, the APCs can bemodified by exposure to the isolated antigen, alone or as part of amixture, peptide loading, or by genetically modifying the APC to expressa polypeptide that includes one or more epitopes.

[0043] As used herein, “pharmaceutically acceptable salt” refers to asalt that retains the desired biological activity of the parent compoundand does not impart any undesired toxicological effects. Examples ofsuch salts include, but are not limited to, (a) acid addition saltsformed with inorganic acids, for example hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and the like; andsalts formed with organic acids such as, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonicacid; (b) salts with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium,and the like; or (c) salts formed with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine; or (d) combinations of(a) and (b) or (c), e.g., a zinc tannate salt; and the like. Thepreferred acid addition salts are the trifluoroacetate salt and theacetate salt.

[0044] As used herein, “pharmaceutically acceptable carrier” includesany material which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline.

[0045] Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990).

[0046] As used herein, “adjuvant” includes those adjuvants commonly usedin the art to facilitate the stimulation of an immune response. Examplesof adjuvants include, but are not limited to, helper peptide; aluminumsalts such as aluminum hydroxide gel (alum) or aluminum phosphate;Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway,N.J.); AS-2 (Smith-Kline Beecham); QS-21 (Aquilla); MPL or 3d-MPL(Corixa Corporation, Hamilton, Mont.); LEIF; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andquil A; muramyl tripeptide phosphatidyl ethanolamine or animmunostimulating complex, including cytokines (e.g., GM-CSF orinterleukin-2, -7 or -12) and immunostimulatory DNA sequences. In someembodiments, such as with the use of a polynucleotide vaccine, anadjuvant such as a helper peptide or cytokine can be provided via apolynucleotide encoding the adjuvant.

[0047] As used herein, “a” or “an” means at least one, unless clearlyindicated otherwise.

[0048] As used herein, to “prevent” or “protect against” a condition ordisease means to hinder, reduce or delay the onset or progression of thecondition or disease.

[0049] HSV Polypeptides

[0050] In one embodiment, the invention provides an isolated herpessimplex virus (HSV) polypeptide. The polypeptide comprises an ICP0, VP16(U_(L)48), or U_(L)49 protein or a fragment thereof. In one embodiment,the fragment comprises amino acids 92-101, 92-105, 288-307 or 743-751 ofICP0 or a substitutional variant thereof. In other embodiments, thefragment comprises amino acids 185-197, 209-221, 288-307, 430-449 or437-449 of VP16 (U_(L)48) or a substitutional variant thereof. Inanother embodiment, the fragment comprises amino acids 14-22, 21-35,45-59, 47-55, 49-57, 49-63, 105-190, 177-220 or 193-208 of U_(L)49 or asubstitutional variant thereof. The reference to amino acid residues ismade with respect to the proteins of the HSV-2 genome as described in A.Dolan et al., 1998, J. Virol. 72(3):2010-2021. The amino acid sequencesof ICP0, VP16 (U_(L)48), and U_(L)49 are as follows. ICPO amino acidsequence 1 meprpgtssr adpgperppr qtpgtqpaap hawgmlndmq wlassdseeetevqisdddl (SEQ ID NO: 1) 61 hrdstseags tdtemfeagl mdaatpparp paerqgsptpadaqgscggg pvgeeeaeag 121 gggdvcavct deiapplrcq sfpclhpfci pcmktwiplrntcplcntpv aylivgvtas 181 gsfstipivn dprtrveaea avragtavdf iwtgnprtaprslslgghtv ralsptppwp 241 gtddedddla dvdyvppapr raprrgggga gatrgtsqpaatrpappgap rssssggapl 301 ragvgsgsgg gpavaavvpr vaslppaagg graqarrvgedaaaaegrtp parqpraaqe 361 ppivisdspp psprrpagpg plsfvssssa qvssgpgggglpqssgraar praavaprvr 421 sppraaaapv vsasadaagp appavpvdah raprsrmtqaqtdtqaqslg ragatdargs 481 ggpgaeggpg vprgtntpga aphaaegaaa rprkrrgsdsgpaasssass saaprsplap 541 qgvgakraap rrapdsdsgd rghgplapas agaappsaspssqaavaaas sssassssas 601 sssassssas sssassssas sssasssagg aggsvasasgagerretslg praaaprgpr 661 kcarktrhae ggpepgardp apgltrylpi agvssvvalapyvnktvtgd clpvldmetg 721 higayvvlvd qtgnvadllr aaapawsrrt llpeharncvrppdyptppa sewnslwmtp 781 vgnmlfdqgt lvgaldfhgl rsrhpwsreq gapapagdapaghge VP16 (UL48) amino acid sequence 1 mdllvddlfa dadgvspppp rpaggpkntpaapplyatgr lsqaqlmpsp pmpvppaalf (SEQ ID NO: 2) 61 nrllddlgfs agpalctmldtwnedlfsgf ptnadmyrec kflstlpsdv idwgdahvpe 121 rspidirahg dvafptlpatrdelpsyyea maqffrgelr areesyrtvl anfcsalyry 181 lrasvrqlhr qahmrgrnrdlremlrttia dryyretarl arvlflhlyl flsreilwaa 241 yaeqmmrpdl fdglccdleswrqlaclfqp lmfingsltv rgvpvearrl relnhirehl 301 nlplvrsaaa eepgaplttppvlqgnqars sgyfmllira kldsyssvat segesvmreh 361 aysrgrtrnn ygstieglldlpddddapae aglvaprmsf lsagqrprrl sttapitdvs 421 lgdelrldge evdmtpadalddfdlemlgd vespspgmth dpvsygaldv ddfefeqmft 481 damgiddfgg UL 49 aminoacid sequence 1 mtsrrsvksc preaprgthe elyygpvspa dpesprddfr rgagpmrarprgevrflhyd (SEQ ID NO: 3) 61 eagyalyrds ssdddesrdt arprrsasva gshgpgparappppggpvga ggrshappar 121 tpkmtrgapk asatpatdpa rgrrpaqads avlldapaptasgrtktpaq glakklhfst 181 appsptapwt prvagfnkrv fcaavgrlaa tharlaavqlwdmsrphtde dlnelldltt 241 irvtvcegkn llqranelvn pdaaqdvdat aaargrpagraaatarapar sasrprrple

[0051] The polypeptide can be a fusion protein. In one embodiment, thefusion protein is soluble. A soluble fusion protein of the invention canbe suitable for injection into a subject and for eliciting an immuneresponse. Within certain embodiments, a polypeptide can be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence. A fusion partner may, for example, assist inproviding T helper epitopes (an immunological fusion partner),preferably T helper epitopes recognized by humans, or may assist inexpressing the protein (an expression enhancer) at higher yields thanthe native recombinant protein. Certain preferred fusion partners areboth immunological and expression enhancing fusion partners. Otherfusion partners may be selected so as to increase the solubility of theprotein or to enable the protein to be targeted to desired intracellularcompartments. Still further fusion partners include affinity tags, whichfacilitate purification of the protein.

[0052] Fusion proteins may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusion proteinis expressed as a recombinant protein, allowing the production ofincreased levels, relative to a non-fused protein, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion protein that retains the biological activity ofboth component polypeptides.

[0053] A peptide linker sequence may be employed to separate the firstand the second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., 1985, Gene 40:39-46; Murphy et al., 1986, Proc. Natl. Acad. Sci.USA 83:8258-8262; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

[0054] The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located 5′ to the DNAsequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals arepresent 3′ to the DNA sequence encoding the second polypeptide.

[0055] Fusion proteins are also provided that comprise a polypeptide ofthe present invention together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a recallresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, for example, Stoute et al., 1997, New Engl. J.Med., 336:86-9).

[0056] Within preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenza virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

[0057] In another embodiment, the immunological fusion partner is theprotein known as LYTA, or a portion thereof (preferably a C-terminalportion). LYTA is derived from Streptococcus pneumoniae, whichsynthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encodedby the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin thatspecifically degrades certain bonds in the peptidoglycan backbone. TheC-terminal domain of the LYTA protein is responsible for the affinity tothe choline or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

[0058] In some embodiments, it may be desirable to couple a therapeuticagent and a polypeptide of the invention, or to couple more than onepolypeptide of the invention. For example, more than one agent orpolypeptide may be coupled directly to a first polypeptide of theinvention, or linkers that provide multiple sites for attachment can beused. Alternatively, a carrier can be used. Some molecules areparticularly suitable for intercellular trafficking and proteindelivery, including, but not limited to, VP22 (Elliott and O'Hare, 1997,Cell 88:223-233; see also Kim et al., 1997,J. Immunol. 159:1666-1668;Rojas et al., 1998, Nature Biotechnology 16:370; Kato et al., 1998, FEBSLett. 427(2):203-208; Vives et al., 1997, J. Biol. Chem.272(25):16010-7; Nagahara et al., 1998, Nature Med. 4(12):1449-1452).

[0059] A carrier may bear the agents or polypeptides in a variety ofways, including covalent bonding either directly or via a linker group.Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No.4,507,234, to Kato et al.), peptides and polysaccharides such asaminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carriermay also bear an agent by noncovalent bonding or by encapsulation, suchas within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and4,873,088).

[0060] In general, polypeptides (including fusion proteins) andpolynucleotides as described herein are isolated. An “isolated”polypeptide or polynucleotide is one that is removed from its originalenvironment. For example, a naturally occurring protein is isolated ifit is separated from some or all of the coexisting materials in thenatural system. Preferably, such polypeptides are at least about 90%pure, more preferably at least about 95% pure and most preferably atleast about 99% pure. A polynucleotide is considered to be isolated if,for example, it is cloned into a vector that is not part of the naturalenvironment.

[0061] The polypeptide can be isolated from its naturally occurringform, produced by recombinant means or synthesized chemically.Recombinant polypeptides encoded by DNA sequences described herein canbe readily prepared from the DNA sequences using any of a variety ofexpression vectors known to those of ordinary skill in the art.Expression may be achieved in any appropriate host cell that has beentransformed or transfected with an expression vector containing a DNAmolecule that encodes a recombinant polypeptide. Suitable host cellsinclude prokaryotes, yeast and higher eukaryotic cells. Preferably thehost cells employed are E. coli yeast or a mammalian cell line such asCos or CHO. Supernatants from the soluble host/vector systems thatsecrete recombinant protein or polypeptide into culture media may befirst concentrated using a commercially available filter. Followingconcentration, the concentrate may be applied to a suitable purificationmatrix such as an affinity matrix or an ion exchange resin. Finally, oneor more reverse phase HPLC steps can be employed to further purify arecombinant polypeptide.

[0062] Fragments and other variants having less than about 100 aminoacids, and generally less than about 50 amino acids, may also begenerated by synthetic means, using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereinamino acids are sequentially added to a growing amino acid chain(Merrifield, 1963,J. Am. Chem. Soc. 85:2146-2149). Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions.

[0063] Variants of the polypeptide for use in accordance with theinvention can have one or more amino acid substitutions, deletions,additions and/or insertions in the amino acid sequence indicated thatresult in a polypeptide that retains the ability to elicit an immuneresponse to HSV or HSV-infected cells. Such variants may generally beidentified by modifying one of the polypeptide sequences describedherein and evaluating the reactivity of the modified polypeptide using aknown assay such as a T cell assay described herein. Polypeptidevariants preferably exhibit at least about 70%, more preferably at leastabout 90%, and most preferably at least about 95% identity to theidentified polypeptides. These amino acid substitutions include, but arenot necessarily limited to, amino acid substitutions known in the art as“conservative”.

[0064] A “conservative” substitution is one in which an amino acid issubstituted for another amino acid that has similar properties, suchthat one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Amino acid substitutions may generally be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity and/or the amphipathic nature of theresidues. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, set, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

[0065] One can readily confirm the suitability of a particular variantby assaying the ability of the variant polypeptide to elicit an immuneresponse. The ability of the variant to elicit an immune response can becompared to the response elicited by the parent polypeptide assayedunder identical circumstances. One example of an immune response is acellular immune response. The assaying can comprise performing an assaythat measures T cell stimulation or activation. Examples of T cellsinclude CD4 and CD8 T cells.

[0066] One example of a T cell stimulation assay is a cytotoxicityassay, such as that described in Koelle, D M et al., Human Immunol.1997, 53;195-205. In one example, the cytotoxicity assay comprisescontacting a cell that presents the antigenic viral peptide in thecontext of the appropriate HLA molecule with a T cell, and detecting theability of the T cell to kill the antigen presenting cell. Cell killingcan be detected by measuring the release of radioactive ⁵¹Cr from theantigen presenting cell. Release of ⁵¹Cr into the medium from theantigen presenting cell is indicative of cell killing. An exemplarycriterion for increased killing is a statistically significant increasein counts per minute (cpm) based on counting of ⁵¹Cr radiation in mediacollected from antigen presenting cells admixed with T cells as comparedto control media collected from antigen presenting cells admixed withmedia.

[0067] Polynucleotides, Vectors, Host Cells and Recombinant Viruses

[0068] The invention provides polynucleotides that encode one or morepolypeptides of the invention. The complete genome sequence of HSV-2,strain HG52, can be found on the NCBI web site (www.ncbi.nih.gov),Accession No. Z86099. The polynucleotide can be included in a vector.The vector can further comprise an expression control sequence operablylinked to the polynucleotide of the invention. In some embodiments, thevector includes one or more polynucleotides encoding other molecules ofinterest. In one embodiment, the polynucleotide of the invention and anadditional polynucleotide can be linked so as to encode a fusionprotein.

[0069] Within certain embodiments, polynucleotides may be formulated soto permit entry into a cell of a mammal, and expression therein. Suchformulations are particularly useful for therapeutic purposes, asdescribed below. Those of ordinary skill in the art will appreciate thatthere are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employed. For example, apolynucleotide may be incorporated into a viral vector such as, but notlimited to, adenovirus, adeno-associated virus, retrovirus, or vacciniaor other pox virus (e.g., avian pox virus). Techniques for incorporatingDNA into such vectors are well known to those of ordinary skill in theart. A retroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selection oftransduced cells) and/or a targeting moiety, such as a gene that encodesa ligand for a receptor on a specific target cell, to render the vectortarget specific. Targeting may also be accomplished using an antibody,by methods known to those of ordinary skill in the art.

[0070] The invention also provides a host cell transformed with a vectorof the invention. The transformed host cell can be used in a method ofproducing a polypeptide of the invention. The method comprises culturingthe host cell and recovering the polypeptide so produced. The recoveredpolypeptide can be purified from culture supernatant.

[0071] Vectors of the invention can be used to genetically modify acell, either in vivo, ex vivo or in vitro. Several ways of geneticallymodifying cells are known, including transduction or infection with aviral vector either directly or via a retroviral producer cell, calciumphosphate precipitation, fusion of the recipient cells with bacterialprotoplasts containing the DNA, treatment of the recipient cells withliposomes or microspheres containing the DNA, DEAE dextran,receptor-mediated endocytosis, electroporation, micro-injection, andmany other techniques known to those of skill. See, e.g., Sambrook etal. Molecular Cloning—A Laboratory Manual (2nd ed.) 1-3, 1989; andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement).

[0072] Examples of viral vectors include, but are not limited toretroviral vectors based on, e.g., HIV, SIV, and murine retroviruses,gibbon ape leukemia virus and other viruses such as adeno-associatedviruses (AAVs) and adenoviruses. (Miller et al. 1990, Mol. Cell Biol.10:4239;J. Kolberg 1992, NIH Res. 4:43, and Cornetta et al. 1991, Hum.Gene Ther. 2:215). Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),ecotropic retroviruses, simian immunodeficiency virus (SIV), humanimmunodeficiency virus (HIV), and combinations. See, e.g. Buchscher etal. 1992, J. Virol. 66(5):2731-2739; Johann et al. 1992, J. Virol.66(5):1635-1640; Sommerfelt et al. 1990, Virol. 176:58-59; Wilson et al.1989,J. Virol. 63:2374-2378; Miller et al. 1991,J. Virol. 65:2220-2224,and Rosenberg and Fauci 1993 in Fundamental Immunology, Third Edition,W. E. Paul (ed.) Raven Press, Ltd., New York and the references therein;Miller et al. 1990, Mol. Cell. Biol. 10:4239; R. Kolberg 1992,J. NIHRes. 4:43; and Cornetta et al. 1991, Hum. Gene Ther. 2:215.

[0073] In vitro amplification techniques suitable for amplifyingsequences to be subcloned into an expression vector are known. Examplesof such in vitro amplification methods, including the polymerase chainreaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplificationand other RNA polymerase mediated techniques (e.g., NASBA), are found inSambrook et al. 1989, Molecular Cloning—A Laboratory Manual (2nd Ed)1-3; and U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds.) Academic Press Inc. San Diego, Calif.1990. Improved methods of cloning in vitro amplified nucleic acids aredescribed in U.S. Pat. No. 5,426,039.

[0074] The invention additionally provides a recombinant microorganismgenetically modified to express a polynucleotide of the invention. Therecombinant microorganism can be useful as a vaccine, and can beprepared using techniques known in the art for the preparation of liveattenuated vaccines. Examples of microorganisms for use as live vaccinesinclude, but are not limited to, viruses and bacteria. In a preferredembodiment, the recombinant microorganism is a virus. Examples ofsuitable viruses include, but are not limited to, vaccinia virus, canarypox virus, retrovirus, lentivirus, HSV and adenovirus.

[0075] Compositions

[0076] The invention provides compositions that are useful for treatingand preventing HSV infection. The compositions can be used to inhibitviral replication and to kill virally-infected cells. In one embodiment,the composition is a pharmaceutical composition. The composition cancomprise a therapeutically or prophylactically effective amount of apolypeptide, polynucleotide, recombinant virus, APC or immune cell ofthe invention. An effective amount is an amount sufficient to elicit oraugment an immune response, e.g., by activating T cells. One measure ofthe activation of T cells is a cytotoxicity assay, as described in D. M.Koelle et al., 1997, Human Immunol. 53:195-205. In some embodiments, thecomposition is a vaccine.

[0077] The composition can optionally include a carrier, such as apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions of the present invention.Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, and carriersinclude aqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, preservatives,liposomes, microspheres and emulsions.

[0078] The composition of the invention can further comprise one or moreadjuvants. Examples of adjuvants include, but are not limited to, helperpeptide, alum, Freund's, muramyl tripeptide phosphatidyl ethanolarine oran immunostimulating complex, including cytokines. In some embodiments,such as with the use of a polynucleotide vaccine, an adjuvant such as ahelper peptide or cytokine can be provided via a polynucleotide encodingthe adjuvant. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Pharmaceuticalcompositions and vaccines within the scope of the present invention mayalso contain other compounds, which may be biologically active orinactive. For example, one or more immunogenic portions of other viralantigens may be present, either incorporated into a fusion polypeptideor as a separate compound, within the composition or vaccine.

[0079] A pharmaceutical composition or vaccine may contain DNA encodingone or more of the polypeptides of the invention, such that thepolypeptide is generated in situ. As noted above, the DNA may be presentwithin any of a variety of delivery systems known to those of ordinaryskill in the art, including nucleic acid expression systems, bacteriaand viral expression systems. Numerous gene delivery techniques are wellknown in the art, such as those described by Rolland, 1998, Crit. Rev.Therap. Drug Carrier Systems 15:143-198, and references cited therein.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) thatexpresses an immunogenic portion of the polypeptide on its cell surfaceor secretes such an epitope. In a preferred embodiment, the DNA may beintroduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc.Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. My Acad.Sci. 569:86-103; Flexner et al., 1990, Vaccine 8:17-21; U.S. Pat.Nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91102805; Berkner, 1988,Biotechniques 6:616-627; Rosenfeld et al., 1991, Science 252:431-434;Kolls et al., 1994, Proc. Natl. Acad. Sci. USA 91:215-219; Kass-Eisleret al., 1993, Proc. Natl. Acad. Sci. USA 90:11498-11502; Guzman et al.,1993, Circulation 88:2838-2848; and Guzman et al., 1993, Cir. Res.73:1202-1207. Techniques for incorporating DNA into such expressionsystems are well known to those of ordinary skill in the art. The DNAmay also be “naked,” as described, for example, in Ulmer et al., 1993,Science 259:1745-1749 and reviewed by Cohen, 1993, Science259:1691-1692. The uptake of naked DNA may be increased by coating theDNA onto biodegradable beads, which are efficiently transported into thecells.

[0080] While any suitable carrier known to those of ordinary skill inthe art may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, intravenous, intracranial, intraperitoneal,subcutaneous or intramuscular administration. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactate polyglycolate)may also be employed as carriers for the pharmaceutical compositions ofthis invention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

[0081] Such compositions may also comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate. Compounds may also be encapsulatedwithin liposomes using well known technology.

[0082] Any of a variety of adjuvants may be employed in the vaccines ofthis invention. Most adjuvants contain a substance designed to protectthe antigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapeussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such as GMCSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0083] Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6, IL-10 and TNF-β) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, 1989, Ann.Rev. Immunol. 7:145-173.

[0084] Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL™ adjuvants are available from CorixaCorporation (see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) also induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 96/02555. Another preferred adjuvant is a saponin, preferably QS21,which may be used alone or in combination with other adjuvants. Forexample, an enhanced system involves the combination of a monophosphoryllipid A and saponin derivative, such as the combination of QS21 and3D-MPL as described in WO 94/00153, or a less reactogenic compositionwhere the QS21 is quenched with cholesterol, as described in WO96/33739. Other preferred formulations comprises an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210. Another adjuvant that may be used is AS-2(Smith-Kline Beecham). Any vaccine provided herein may be prepared usingwell known methods that result in a combination of antigen, immuneresponse enhancer and a suitable carrier or excipient.

[0085] The compositions described herein may be administered as part ofa sustained release formulation (i.e., a formulation such as a capsuleor sponge that effects a slow release of compound followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. The amount of activecompound contained within a sustained release formulation depends uponthe site of implantation, the rate and expected duration of release andthe nature of the condition to be treated or prevented.

[0086] Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets HSV-infected cells.Delivery vehicles include antigen presenting cells (APCs), such asdendritic cells, macrophages, B cells, monocytes and other cells thatmay be engineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have antiviral effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

[0087] Certain preferred embodiments of the present invention usedendritic cells or progenitors thereof as antigen-presenting cells.Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticimmunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). Ingeneral, dendritic cells may be identified based on their typical shape(stellate in situ, with marked cytoplasmic processes (dendrites) visiblein vitro) and based on the lack of differentiation markers of B cells(CD19 and CD20), T cells (CD3), monocytes (CD14) and natural killercells (CD56), as determined using standard assays. Dendritic cells may,of course, be engineered to express specific cell-surface receptors orligands that are not commonly found on dendritic cells in vivo or exvivo, and such modified dendritic cells are contemplated by the presentinvention. As an alternative to dendritic cells, secreted vesiclesantigen-loaded dendritic cells (called exosomes) may be used within avaccine (Zitvogel et al., 1998, Nature Med. 4:594-600).

[0088] Dendritic cells and progenitors may be obtained from peripheralblood, bone marrow, tumor-infiltrating cells, peritumoraltissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cordblood or any other suitable tissue or fluid. For example, dendriticcells may be differentiated ex vivo by adding a combination of cytokinessuch as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytesharvested from peripheral blood. Alternatively, CD34 positive cellsharvested from peripheral blood, umbilical cord blood or bone marrow maybe differentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce maturation and proliferation of dendriticcells.

[0089] Dendritic cells are conveniently categorized as “immature” andmature” cells, which allows a simple way to discriminate between twowell-characterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor, mannose receptor and DEC-205marker. The mature phenotype is typically characterized by a lowerexpression of these markers, but a high expression of cell surfacemolecules responsible for T cell activation such as class I and class IIMHC, adhesion molecules (e.g., CD54 and CD11) and costimulatorymolecules (e.g., CD40, CD80 and CD86). APCs may generally be transfectedwith a polynucleotide encoding a polypeptide (or portion or othervariant thereof) such that the polypeptide, or an immunogenic portionthereof, is expressed on the cell surface. Such transfection may takeplace ex vivo, and a composition or vaccine comprising such transfectedcells may then be used for therapeutic purposes, as described herein.Alternatively, a gene delivery vehicle that targets a dendritic or otherantigen presenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo and ex vivo transfection ofdendritic cells, for example, may generally be performed using anymethods known in the art, such as those described in WO 97/24447, or thegene gun approach described by Mahvi et al., 1997, Immunology and CellBiology 75:456-460. Antigen loading of dendritic cells may be achievedby incubating dendritic cells or progenitor cells with the tumorpolypeptide, DNA (naked or within a plasmid vector) or RNA; or withantigen-expressing recombinant bacterium or viruses (e.g., vaccinia,fowlpox, adenovirus or lentivirus vectors). Prior to loading, thepolypeptide may be covalently conjugated to an immunological partnerthat provides T cell help (e.g., a carrier molecule). Alternatively, adendritic cell may be pulsed with a non-conjugated immunologicalpartner, separately or in the presence of the polypeptide.

[0090] Administration of the Compositions

[0091] Treatment includes prophylaxis and therapy. Prophylaxis ortreatment can be accomplished by a single direct injection at a singletime point or multiple time points. Administration can also be nearlysimultaneous to multiple sites. Patients or subjects include mammals,such as human, bovine, equine, canine, feline, porcine, and ovineanimals. Preferably, the patients or subjects are human.

[0092] Compositions are typically administered in vivo via parenteral(e.g. intravenous, subcutaneous, and intramuscular) or other traditionaldirect routes, such as buccal/sublingual, rectal, oral, nasal, topical,(such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial,intraperitoneal, intraocular, or intranasal routes or directly into aspecific tissue.

[0093] The compositions are administered in any suitable manner, oftenwith pharmaceutically acceptable carriers. Suitable methods ofadministering cells in the context of the present invention to a patientare available, and, although more than one route can be used toadminister a particular cell composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

[0094] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time, or to inhibit infection or diseasedue to infection. Thus, the composition is administered to a patient inan amount sufficient to elicit an effective immune response to thespecific antigens and/or to alleviate, reduce, cure or at leastpartially arrest symptoms and/or complications from the disease orinfection. An amount adequate to accomplish this is defined as a“therapeutically effective dose.”

[0095] The dose will be determined by the activity of the compositionproduced and the condition of the patient, as well as the body weight orsurface areas of the patient to be treated. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside effects that accompany the administration of a particularcomposition in a particular patient. In determining the effective amountof the composition to be administered in the treatment or prophylaxis ofdiseases such as HSV infection, the physician needs to evaluate theproduction of an immune response against the virus, progression of thedisease, and any treatment-related toxicity.

[0096] For example, a vaccine or other composition containing a subunitHSV protein can include 1-10,000 micrograms of HSV protein per dose. Ina preferred embodiment, 10-1000 micrograms of HSV protein is included ineach dose in a more preferred embodiment 10-100 micrograms of HSVprotein dose. Preferably, a dosage is selected such that a single dosewill suffice or, alternatively, several doses are administered over thecourse of several months. For compositions containing HSVpolynucleotides or peptides, similar quantities are administered perdose.

[0097] In one embodiment, between 1 and 10 doses may be administeredover a 52 week period. Preferably, 6 doses are administered, atintervals of 1 month, and booster vaccinations may be given periodicallythereafter. Alternate protocols may be appropriate for individualpatients. A suitable dose is an amount of a compound that, whenadministered as described above, is capable of promoting an antiviralimmune response, and is at least 10-50% above the basal (i.e.,untreated) level. Such vaccines should also be capable of causing animmune response that leads to an improved clinical outcome in vaccinatedpatients as compared to non-vaccinated patients. In general, forpharmaceutical compositions and vaccines comprising one or morepolypeptides, the amount of each polypeptide present in a dose rangesfrom about 0.1 μg to about 5 mg per kg of host. Preferably, the amountranges from about 10 to about 1000 μg per dose. Suitable volumes foradministration will vary with the size, age and immune status of thepatient, but will typically range from about 0.1 mL to about 5 mL, withvolumes less than about 1 mL being most common.

[0098] Compositions comprising immune cells are preferably prepared fromimmune cells obtained from the subject to whom the composition will beadministered. Alternatively, the immune cells can be prepared from anHLA-compatible donor. The immune cells are obtained from the subject ordonor using conventional techniques known in the art, exposed to APCsmodified to present an epitope of the invention, expanded ex vivo, andadministered to the subject. Protocols for ex vivo therapy are describedin Rosenberg et al., 1990, New England J. Med. 9:570-578. In addition,compositions can comprise APCs modified to present an epitope of theinvention.

[0099] Immune cells may generally be obtained in sufficient quantitiesfor adoptive immunotherapy by growth in vitro, as described herein.Culture conditions for expanding single antigen-specific effector cellsto several billion in number with retention of antigen recognition invivo are well known in the art. Such in vitro culture conditionstypically use intermittent stimulation with antigen, often in thepresence of cytokines (such as IL-2) and non-dividing feeder cells. Asnoted above, immunoreactive polypeptides as provided herein may be usedto enrich and rapidly expand antigen-specific T cell cultures in orderto generate a sufficient number of cells for immunotherapy. Inparticular, antigen-presenting cells, such as dendritic, macrophage,monocyte, fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., 1997, Immunological Reviews 157:177).

[0100] Administration by many of the routes of administration describedherein or otherwise known in the art may be accomplished simply bydirect administration using a needle, catheter or related device, at asingle time point or at multiple time points.

[0101] In Vivo Testing of Identified Antigens

[0102] Conventional techniques can be used to confirm the in vivoefficacy of the identified HSV antigens. For example, one techniquemakes use of a mouse challenge model. Those skilled in the art, however,will appreciate that these methods are routine, and that other modelscan be used.

[0103] Once a compound or composition to be tested has been prepared,the mouse or other subject is immunized with a series of injections. Forexample up to 10 injections can be administered over the course ofseveral months, typically with one to 4 weeks elapsing between doses.Following the last injection of the series, the subject is challengedwith a dose of virus established to be a uniformly lethal dose. Acontrol group receives placebo, while the experimental group is activelyvaccinated. Alternatively, a study can be designed using sublethaldoses. Optionally, a dose-response study can be included. The end pointsto be measured in this study include death and severe neurologicalimpairment, as evidenced, for example, by spinal cord gait. Survivorscan also be sacrificed for quantitative viral cultures of key organsincluding spinal cord, brain, and the site of injection. The quantity ofvirus present in ground up tissue samples can be measured. Compositionscan also be tested in previously infected animals for reduction inrecurrence to confirm efficacy as a therapeutic vaccine.

[0104] Efficacy can be determined by calculating the IC₅₀, whichindicates the micrograms of vaccine per kilogram body weight requiredfor protection of 50% of subjects from death. The IC₅₀ will depend onthe challenge dose employed. In addition, one can calculate the LD₅₀,indicating how many infectious units are required to kill one half ofthe subjects receiving a particular dose of vaccine. Determination ofthe post mortem viral titer provides confirmation that viral replicationwas limited by the immune system.

[0105] A subsequent stage of testing would be a vaginal inoculationchallenge. For acute protection studies, mice can be used. Because theycan be studied for both acute protection and prevention of recurrence,guinea pigs provide a more physiologically relevant subject forextrapolation to humans. In this type of challenge, a non-lethal dose isadministered, the guinea pig subjects develop lesions that heal andrecur. Measures can include both acute disease amelioration andrecurrence of lesions. The intervention with vaccine or othercomposition can be provided before or after the inoculation, dependingon whether one wishes to study prevention versus therapy.

[0106] Methods

[0107] The invention provides a method for treatment and/or preventionof HSV infection in a subject. The method comprises administering to thesubject a composition of the invention. The composition can be used as atherapeutic or prophylactic vaccine. In one embodiment, the HSV isHSV-2. Alternatively, the HSV is HSV-1. The invention additionallyprovides a method for inhibiting HSV replication, for killingHSV-infected cells, for increasing secretion of lymphokines havingantiviral and/or immunomodulatory activity, and for enhancing productionof herpes-specific antibodies. The method comprises contacting anHSV-infected cell with an immune cell directed against an antigen of theinvention, for example, as described in the Examples presented herein.The contacting can be performed in vitro or in vivo. In a preferredembodiment, the immune cell is a T cell. T cells include CD4 and CD8 Tcells. Compositions of the invention can also be used as a tolerizingagent against immunopathologic disease.

[0108] In addition, the invention provides a method of producing immunecells directed against HSV. The method comprises contacting an immunecell with an HSV polypeptide of the invention. The immune cell can becontacted with the polypeptide via an antigen-presenting cell, whereinthe antigen-presenting cell is modified to present an antigen includedin a polypeptide of the invention. Preferably, the antigen-presentingcell is a dendritic cell. The cell can be modified by, for example,peptide loading or genetic modification with a nucleic acid sequenceencoding the polypeptide. In one embodiment, the immune cell is a Tcell. T cells include CD4 and CD8 T cells. Also provided are immunecells produced by the method. The immune cells can be used to inhibitHSV replication, to kill HSV-infected cells, in vitro or in vivo, toincrease secretion of lymphokines having antiviral and/orimmunomodulatory activity, to enhance production of herpes-specificantibodies, or in the treatment or prevention of HSV infection in asubject.

[0109] The invention also provides a diagnostic assay. The diagnosticassay can be used to identify the immunological responsiveness of apatient suspected of having a herpetic infection and to predictresponsiveness of a subject to a particular course of therapy. The assaycomprises exposing T cells of a subject to an antigen of the invention,in the context of an appropriate APC, and testing for immunoreactivityby, for example, measuring IFNγ, proliferation or cytotoxicity. Suitableassays are described in more detail in the Examples.

EXAMPLES

[0110] The following examples are presented to illustrate the presentinvention and to assist one of ordinary skill in making and using thesame. The examples are not intended in any way to otherwise limit thescope of the invention.

Example 1 Identification of Viral Epitopes in HSV-2 Tegument Proteins

[0111] This example shows the use of expression cloning with full-lengthviral DNA to identify T-cell antigens. Described herein are HSV epitopesrecognized by lesion-infiltrating T-cells discovered by expressioncloning. Details of the methods are described in U.S. Pat. No.6,375,952, issued Apr. 23, 2002.

[0112] Lymphocyte Functional Assays

[0113] Triplicate proliferation assay wells contained 10⁴ clonedT-cells, 10⁵ irradiated (3300 rad) PBMC or 2.5×10⁴ irradiated (8000 rad)EBV-LCL as antigen presenting cells (APC), and antigen in 200 μl T-cellmedia (D. M. Koelle et al., 1997, Human. Immunol., 53:195-205) in96-well U-bottom plates. When heat-killed bacteria were used as antigen,the equivalent of 10⁵ cfu/well (prior to inactivation) was added andgentamicin (20 μg/ml) was included. After 72 hours, 1 μCi/well [³]Hthymidine was added for 18 hours, cells were harvested, andincorporation of thymidine evaluated by liquid scintillation counting.Standard deviations were less than 10% of the mean values. Results arereported as mean cpm or as delta cpm=mean cpm for experimental antigenminus mean cpm for control antigen. Control antigen was mock-infectedcell lysate for whole viral antigens and pUEX2-derived β-galactosidasefor recombinant protein preparations. To determine the reactivity ofbulk-cultured lesion-derived T-cells, fusion proteins or controlβ-galactosidase were used at 10 μg/ml. To determine HLA restrictingloci, HLA DR-specific mAb L243 (V. G. Preston et al., 1978, J. Virol.,28:499-517), HLA DP-specific mAb B7.21 (A. J. Watson et al., 1983,Nature, 304:358-360), or HLA DQ-specific mAb SpV-L3 (H. Spits et al.,1984, Eur. J. Immunol., 14:299-304) were used as described (D. M. Koelleet al., 1994,J. Virol. 68:2803-2810).

[0114] Cytolysis assays were performed in triplicate using 4-hour [⁵¹]Crrelease as described (D. M. Koelle et al., 1993,J. Clin. Invest.,91:961-968). Target EBV-LCL were infected for 18 hours with HSV-3 at amultiplicity of infection of 30 or pulsed with 1.0 μM peptide for 90minutes prior to washing as described (W. W. Kwok et al., 1996,J. Exp.Med., 183:1253-1258). The effector to target ratio was 20:1. Spontaneousrelease was less than 28%.

[0115] Results

[0116] Fine Localization of T-Cell Epitopes

[0117] To reduce the complexity of libraries for expression cloning, TCCrecognizing antigen(s) partially mapped using HSV-1 X HSV-2 intertypicrecombinant viruses (IRV) were selected. HSV DNA near 0.7 map unitesencodes T-cell antigens in addition to VP16. Epitope mapping for TCC4.2EI and 2.3 (D. M. Koelle et al., 1994, J. Virol., 68:2803-2810) wasimproved with IRV DX32 (FIG. 1A). This HSV-2 based virus contains ablock of HSV-1 DNA near 0.7 map units (V. G. Preston et al., 1978,J.Virol., 28:499-517). The U_(L)48 gene product has the HSV-2 phenotype,as shown by reactivity with HSV-2 type-specific, VP16-specific (D. M.Koelle et al., 1994,J. Virol., 68:2803-2810) T-cell clone 1A.B.25. TheU_(L)49 (FIG. 2) and U_(L)50 gene products (M. V. Williams, 1987,Virology, 156:282-292; F. Wohlrab, 1982,J. Virol., 43:935-942) also havea HSV-2 phenotype. The HSV-2 DNA present in IRV DX32 therefore includesU_(L)48, U_(L)49, U_(L)50, and most likely the intervening U_(L)^(49.5). Since TCC 4.2E1 and 2.3 react with RS1G31 and DX32, but notwith RP2 (FIG. 1B), recognition of U_(L)49, U_(L)49.5, or U_(L)50 ismost likely.

[0118] Expression Cloning to Determine T-Cell Antigens

[0119] The BamH I w fragment of HSV-2 was selected for expressioncloning, since it contains the U_(L)49, U_(L)49.5, and most of theU_(L)50 coding sequences (A. Cress and S. J. Triezenberg, 1991, Gene,103:235-238; G. D. Elliott and D. M. Meredith, 1992, J. Gen. Virol.,73:723-736; N. J. Maitland et al., 1982, Infect. Immun., 38:834-842).70-90% of random colonies contained an insert; all were of viral origin.The most active libraries (Table 1) for each TCC (pUEX1 for TCC 4.2E1,pUEX 3 for TCC 2.3) were selected and an individual reactive bacterialclone detected by sequential testing of pools and individual colonies(Table 2). Clone 1.1.3 encodes a fusion protein eliciting proliferationby TCC 4.2E1. This clone contains a backwards 80 bp Sma I fragment ofU_(L)49, a 262 bp Sma I fragment of HSV-2 U_(L)49 DNA predicted toencode amino acids 105 to 190, forward and in-frame with regards toβ-galactosidase, and a 246 bp Sma I fragment of U_(L)49 forward but outof frame due to a deletion of a single C residue at the 262 bp Sma Ifragment-242 bp Sma I fragment junction. Clone 3.19 contained a 583 bpSma I fragment encoding amino acids 118-312 of U_(L)50, followed bybackwards 80 and 96 bp Sma I fragments of U_(L)49. TABLE 1Identification of protein libraries eliciting proliferation (mean cpm[³H] thymidine incorporation) of HSV-specific TCC. Autologous EBV-LCL(clones 4.2E1 and 2.3) or PBMC were used as APC and library-derivedfusion protein antigens were diluted 1:300. Data are mean cpm [³H]thymidine incorporation. library¹ pUEX1-BamHI pUEX2-BamHI pUEX3-BamHIcontrol stimuli² TCC “w”-SmaI “w”-SmaI “w”-SmaI media HSV-2 4.2E1 10,1054,150 1,903 286 21,591 2.3   418   785 2,279 102 11,014 pUEX1-HG52-pUEX2-HG52- pUEX3-HG52- SmaI-Alu I SmaI-Alu I SmaI-Alu I ESL4.9 −52 −2516,235 146 66,013 ESL2.20 1 768  5,427 123 13,359

[0120] Identification of T-cell antigens was confirmed by targetedsubcloning and overlapping peptides. The 262 bp Sma I fragment ofU_(L)49 of HSV-2 encoding amino acids 105-190 was subcloned into pUEX3to yield plasmid 49.262.12. This protein stimulated TCC 4.2E1 (Table 2).Only peptide 105-126 of VP22 of HSV-2 (GGPVGAGGRSHAPPARTPKMTR; SEQ IDNO: 4) was stimulatory (FIG. 3). DNA fragments encoding U_(L)50 118-312and 118-250 were subcloned into pUEX3. Fusion proteins expressing thesefragments were active (Table 2). TABLE 2 Antigenic specificity of HSV-2reactive TCC. Bacterially-derived recombinant fusion protein antigenswere used at 1:900 dilution. Autologous EBV-LCL (clone 4.2E1) or PBMCwere used as APC. Data are delta cpm [³H] thymidine incorporationcompared to media, which was less than 500 cpm in each case. recombinantantigen Clone control antigens TCC name viral sequence¹ cpm pUEX2 β-galHSV-1 HSV-2 4.2E1 1.1.3 VP22 105-190  4,875  93 nd nd 49.262.12² VP22105-190  6,898 2.3 3.19 U_(L)50 118-312 43,971 231    543 53,03250.583.44³ U_(L)50 118-312 34,453 50.397³ U_(L)50 118-250 66,501 ESL4.9C11 VP22 177-220 59,400 166 112,803 64,685

[0121] Evaluation of random colonies from full-length HSV-2 DNAlibraries showed that 80-100% contained plasmids with an insert; 80-100%of inserts were of viral origin. For both TCC ESL4.9 and ESL2.20, onlythe pUEX3 protein library elicited lymphoproliferation (Table 1). Sincethe libraries were more complex than for those made from the BamH I wfragment, 2,000-3,000 bacterial transformants were screened by acombinatorial method. In preliminary experiments, heat-killed, washedbacteria were found to substitute for inclusion body preparations ofprotein in lymphoproliferation assays at the pool (5-12 bacterialclones) and final assay stages.

[0122] Sequencing of plasmids in positive bacteria showed that TCCESL4.9 recognized a 44 amino acid fragment of U_(L)49 gene product VP22(amino acids 177-220), while TCC ESL2.20 recognized a 34 amino acidfragment of U_(L)21 (amino acids 148-181) (Table 2). In both casessingle Alu I fragments of HSV-2 DNA were inserted in-frame and forwards.Peptide mapping revealed that amino acids 187-206 (FIG. 3C) stimulatedTCC ESL4.9.

[0123] Fusion Proteins as Probes of Bulk Lesion-Infiltrating T-Cells

[0124] Newly discovered T-cell antigens were added to the panel of HSVantigens used to probe bulk cultures of lesion-infiltrating T-cells. Thefirst available specimens were a set of four biopsies (2 mm each)obtained from day 5 (virus culture positive) of a buttock recurrence ofHSV-2 from patient 1 (D. M. Koelle et al., 1998,J. Clin. Invest.101:1500-09; D. M. Koelle et al., 1994, J. Virol., 68:2803-2810). Allfour biopsies showed reactivity with VP22 105-190 but notβ-galactosidase, glycoproteins B or D, or VP16. TCC were derived afterrestimulating the original bulk culture for one cycle with VP22 105-190fusion protein. Proliferative responses of TCC 1.L3D5.10.8 (FIG. 3B) toVP22 (105-190) and constituent peptides document a third T-cell epitopein VP22 contained within amino acids 125-146.

[0125] HLA Restriction

[0126] The HLA restriction of the TCC recognizing antigens encoded near0.7 map units was determined in detail. Proliferation of TCC 4.2E1,specific for VP22 105-126, is inhibited 84% by anti-DP, but less than20% by anti-DR or anti-DQ niAb. TCC 4.2E1 is from a DPB1*2001/DPB1*0402heterozygous donor. Allogeneic EBV-LCL bearing DPB1*2001, but notDPB1*0402, present antigen (Table 3), establishing restriction byDPB1*2001. Proliferation of TCC 2.3, specific for U_(L)50, was inhibitedby anti-DR but not anti-DP or anti-DQ mAb. This clone is from aDRB1*0301/BRB1*0701 heterozygous donor.

[0127] Allogeneic PBMC from a DRB1*0301 donor presented antigen,consistent with binding of antigenic peptide to this allele. However,presentation by the linked DR gene products DRw52 or DRw53, cannot beruled out. Additional HLA restriction studies are summarized in Table 4.TABLE 3 Determination of restricting HLA allele of lesion-derived CD4TCC 4.2E1 and 2.3. Antigens were β-gal fusion proteins (Table 2) at1:900 dilution. Data are delta cpm [³H] thymidine incorporation comparedto media, which was less than 500 cpm in each case. T-cell clone antigenAPC HLA type¹ delta cpm² 4.2E1 1.1.3 autologous EBV- DPB1*0402, 200130,719 LCL AMAI EBV-LCL DPB1*0402  2,732 ARENT EBV-LCL DPB1*2001 26,2182.3 50.583.44 autologous PBMC DRB1*0301, 0701  8,964 allogeneic PBMC ADRB1*0701, 1001 45 allogeneic PBMC B DRB1*0301, 1301 19,223

[0128] TABLE 4 Cytolytic activity of lesion-derived, tegument-specificCD4 TCC with summary of fine specificity and HLA restriction. Resultsare percent specific release at an effector to target ratio of 20:1except ESL4.34 (10:1). Auto = autologous EBV-LCL as target cells; allo =allogeneic EBV-LCL mismatched at the relevant HLA locus (if known) ormismatch at HLA DR and DQ. cytolysis assay target HLA auto auto autoallo allo allo TCC specificity¹ restriction² HSV-2 peptide mock HSV-2peptide mock 4.2E1 VP22 105- DPB1*2001 20.7 44.2 −4.1 −2.9 −1.7 4.6 126ESL4.9 VP22 187- DR³ −0.6 20.2 1.3 0 0 0 206 1.L3D5.10.8 VP22 125- DR⁴1.1 61.1 −0.3 −0.4 −0.6 −0.4 146 1.L3D5.10.12 VP22 125- DR⁴ 2.5 57.6 1.6−0.1 −2.5 −1.4 146

[0129] The HLA restriction of TCC BM.17 was studied in detail.Proliferation of TCC BM.17 and the similar clone SB.17 was inhibited 90%by anti-DQ, but less than 25% by anti-DR or anti-DP mAb. Donors BM andSB are heterozygous for HLA DQB1*0201/0501. At high concentrations ofpeptide, both DQB1*0201- and DQB1*0501 homozygous EBV-LCL appeared topresent antigen to TCC BM.17.

[0130] CTL Activiy of Tegument-Specific CD4 T-Cell Clones

[0131] Cytotoxic activities of the CD4 TCC with newly and previouslyidentified specificities were tested using EBV-LCL target cells (Table4). All clones tested displayed cytolytic activity towardspeptide-loaded target cells. Cytolytic activity against target cellsinfected with HSV-2 showed greater variability. VP22-specific TCC 4.2E1was active, while VP22-specific TCC from other donors were not.

[0132] Discussion

[0133] HSV-specific T-cells selectively infiltrate recurrent genitalHSV-2 lesions (D. M. Koelle et al., 1994,J. Infect. Dis., 169:956-961).Local CTL activity, with CD4 and CD8-mediated components, is correlatedwith viral clearance D. M. Koelle et al., 1998, J. Clin. Invest.101:1500-09). The antigens recognized by local HSV-specific T cells arediverse and in many cases unknown (D. M. Koelle et al., 1994,J. Virol.,68:2803-2810). This example documents recognition of tegument proteinVP22.

[0134] The expression cloning system described herein works well withHSV. Genomic double stranded DNA can be used directly since introns arerare in the HSV genome. The same HSV-2 strain, HG52 (A. Dolan et al.,1998,J. Virol. 72:2010-2021) was used to screen candidate lesion-derivedTCC and make protein libraries. The relatively low degree of strainvariability (M. J. Novotny et al., 1996, Virology, 221:1-13) betweenHSV-2 strains in the donors and HG52 might rarely lead to omission ofepitope(s) recognized in vivo; application to viruses with more strainvariation would benefit from the use of autologous isolates.

[0135] Notably, reactivity with VP22 was detected in two independentexpression cloning experiments with lesion-infiltrating TCC from twodonors. VP22 reactivity was also detected during screening of the firstavailable set of bulk lesion-infiltrating lymphocyte cultures. Tenadditional clones from three patients have been negative with thedisclosed fragments of U_(L)49, U_(L)21, and U_(L)50.

[0136] Tegument antigens may be suitable targets for lesion-infiltratingCD4 T-cells because of their abundance. VP16 and VP22 are present inlarge amounts: on the order of 1.6×10³ molecules of VP16 (Y. Zhang andJ. L. C. McKnight, 1993, J. Virol., 67:1482-1492) and 2.5-2.8×10³molecules of VP22 (J. Leslie et al., 1996, Virology, 220:60-68) areincorporated into each virion in HSV-1.

[0137] Because polypeptides expressed as C-terminal fusion to VP22 canbe co-transported into cells, expression of proteins as VP22 fusions maybe of interest as a type of adjuvant preparation. This can be tested byexpression of heterologous epitopes in VP22. VP16 and VP22 of HSV-1 arestrongly, noncovalenty associated in infected cells as shown bycommunoprecipitation. These proteins co-localize in the perinuclear areaof cells (G. Elliott et al., 1995,J. Virol., 69:7932-7941; G. D. Elliottet al., 1992,J. Gen. Virol., 73:723-736).

[0138] In summary, expression cloning has allowed discovery of novel HSVT-cell antigens. The in situ enrichment of antigen-specific CD4 T-cellsin lesions allows study of the antigenic repertoire unbiased bysecondary in vitro stimulation with antigen. The favorablecharacteristics of the HSV genome allow direct use of libraries of wholeviral DNA. Tegument proteins are candidates together with membraneglycoproteins for use as HSV vaccines in humans.

Example 2 Efficacy of Full-length U_(L)49

[0139] This Example shows that the full-length U_(L)49 protein iseffective at stimulating T cell proliferation. The data demonstrate theantigenicity of full-length U_(L)49 expressed in E. coli and in Cos-7cells. These results confirm that the antigens described hereinabovewere accurately identified.

[0140] To express full-length U_(L) 49 protein of HSV-2 in a prokaryoticsystem, the gene was cloned by PCR from DNA prepared from HSV type 2strain HG52 using primers GGAAGATCTACCTCTCGCCGCTCCGTCA (SEQ ID NO: 5) atthe 5′ end of the gene and CCGGAATTCTTGTCTGTCGTCTGAACGCG (SEQ ID NO: 6)at the 3′ end of the gene. PCR product was digested with Bgl II and EcoRI and cloned into the Bgl II and EcoR I sites in the TA cloning vectorpcR2.1-Topo (Invitrogen). The gene was then subcloned into the vectorpTrcHisB (Invitrogen) and then into pGEX-2T (Pharmacia). The sequence ofthe HSV-2 U_(L)49 clone had one coding mutation compared to thepublished sequence (Dolan 1998): amino acid 244 was mutated from serineto proline. The predicted amino acid sequence of the expressed proteinalso is missing the initial methionine. U_(L)49 contains an N-terminalfusion domain derived from vector pGEX2T. This plasmid is namedpGEX2T-U_(L)49HSV2.

[0141] To make prokaryotically expressed full length U_(L)49 of HSV-2,pGEX2TU-L49HSV2 or control empty vector was transformed into E. colistrain BL21 Bacteria in log-phase growth were adjusted to an OD₆₀₀ of0.4 in LB-ampicillin media. To some tubes isopropylbeta-D-thiogalactopyranoside (IPTG) was added to 0.3 mM. Bacteria werecultured for 1.5 hours at 37° C. with rotation. Bacteria were collectedby centrifugation and washed 3× in PBS containing 1 mM EDTA, heated to65° C. for 10 minutes, and washed twice more with PBS, and resuspendedat approximately 1×10⁹ bacteria/ml in T-cell medium. Heat-killedbacterial suspensions were used as test antigen.

[0142] To express full-length U_(L)49 protein of HSV-2 in a eukaryoticsystem, the gene was separately re-amplified by polymerase chainreaction using a high-fidelity DNA polymerase with proof-readingfunction. The same primers and template were used. The gene was cloneddirectly into the Bgl II and EcoR I sites of pEGFP-C1 (Clontech). Theentire U_(L)49 gene was sequenced and agreed with published sequence.The predicted amino acid sequence of the expressed protein is identicalto that predicted for viral U_(L)49 except that the initial methionineat amino acid 1 is missing. A N-terminal fusion domain derived fromvector pEGFP-C1 is also predicted to be expressed. This plasmid is namedpEGFP-C1-UL49HSV2.

[0143] To make eukaryotically expressed full length U_(L)49 of HSV-2,pEGFP-C1-UL49HSV2 plasmid DNA or pEGFP-C1vector control DNA wastransfected into Cos-7 cells by lipofection. After 48 hours, cells werescraped and sonicated and a supernatant and pellet phase prepared. Cellsfrom a 9.4 cm² dish were used to prepare 300 microliters of supernatant.The pellet from a 9.4 cm² dish was resuspended in 300 microlitersmedium. Supernatant and pellet preparations were used as test antigens.

[0144] These test antigens were added to assay wells (96-well, U-bottom)in 200 microliters of T-cell medium containing 1×10⁵ autologousirradiated peripheral blood mononuclear cells (PBMC) per well and 1×10⁴lesion-derived CD4-bearing T-cell clone ESL4.9 for U_(L)49 (Koelle etal, 1994 and 1998). Assays were performed in duplicate or triplicate.After three days, ³H thymidine incorporation was measured as describedin Example 1.

[0145] Results are expressed as stimulation index (mean cpm ³H thymidineincorporation test antigen/mean cpm ³H thymidine incorporation mediacontrol) and delta cpm (mean cpm ³H thymidine incorporation test antigenminus mean cpm ³H thymidine incorporation media control). Positive andnegative control antigens were run as indicated and as described inExample 1. TABLE 5 Antigenicity of full-length HSV-2 U_(L) 49 expressedprokaryotically in E. coli BL21 antigen final dilution delta cpmstimulation index UV HSV-2 1:100 26,823 386 heat-killed pGEX2 1:4  −110.84 heat-killed pGEX2 1:40  −25 0.64 heat-killed pGEX2 1:400  −8 0.89heat-killed pGEX2- 1:4   9,413 135 UL49HSV2 heat-killed pGEX2- 1:40 10,526 152 UL49HSV2 heat-killed pGEX2- 1:400  5,021 73 UL49HSV2

[0146] TABLE 6 Antigenicity of full-length HSV-2 U_(L) 49 expressedeukaryotically in Cos-7 cells stimulation antigen final dilution deltaCPM index UV-mock virus 1:100 −4 0.96 UVHSV-2 1:100 46,510 470supernatant of control- 1:4  8 1.08 transfected cells pellet ofcontrol-transfected cells 1:4  131 2.32 supernatant of U_(L)49-transfected 1:4   1,512 16.3 cells pellet of U_(L) 49-transfectedcells 1:4  84,951 859 pellet of U_(L) 49-transfected cells 1:40  35,753362 pellet of U_(L) 49-transfected cells 1:400 29,854 302

[0147] These results show that HSV-2 protein U_(L)49 retains itsimmunogenicity when expressed as a full-length protein. U_(L)49 wasstudied in both prokaryotic and eukaryotic systems.

Example 3 Prevalence of Antigens in Population

[0148] This example supports the utility of preventative and therapeuticuses of the antigens of the invention by demonstrating the prevalence ofresponses to these antigens among the population. To do this, sevenindividuals who were HSV-2 infected as documented by type-specificserology were surveyed. These individuals were different from theindividuals from whom the index T-cell clones were recovered from HSV-2lesions.

[0149] For each subject, PBMC were isolated and plated at 2×10⁶cells/well in 2 mls of T-cell medium in 24-well plates and stimulated invitro with a 1:500 dilution of UV-inactivated HSV-2 strain 333 for fivedays. At that time, 40 units/ml recombinant human IL-2 was added for anadditional five to six days, giving rise to a short-term, HSV-specificcell line termed a B1 cell line.

[0150] Reactivity to individual HSV-2 proteins was assessed as follows.Proliferation assays were set up on 96-well round bottom microtiterplates, and each condition was performed in triplicate. To each well,1×10⁵ autologous irradiated (3300 rad gamma) PBMC were added as antigenpresenting cells. To each well, 1×10⁴ B1 cells were added. The followingcontrol substances were added: media, UV-treated mock virus preparationdiluted 1:500, UV-treated HSV-2 strain 333 diluted 1:500, glycoproteinsB or D or VP16 protein of HSV-2 (purified) at 4 micrograms per ml finalconcentration. The response to UV-treated HSV-2 was expected to bepositive and served as a positive control for the viability and overallspecificity of the cells. Glycoproteins B and D and VP16 were previouslyshown to be targets of HSV-specific T-cells (D. M. Koelle et al.,1994,J. Virol 68(5):2803-2810).

[0151] For the newly discovered antigen UL49, the cloning of thefull-length gene and its expression in the eukaryotic Cos-7 system wasas described above, as was the preparation of control antigens based onthe empty vector. The supernatant and pellet after sonication oftransfected Cos-7 cells was used at a final dilution of 1:20 intriplicate proliferation assays.

[0152] Positive responses were scored if the stimulation index (mean cpm³H thymidine incorporation for test antigen/mean cpm ³H thymidineincorporation for relevant control antigen) was greater than or equal to4.0. For UV HSV-2 antigen, the relevant control antigen was UV-mockvirus. For gB2, gD2, and VP16, the control was media. For the newantigens expressed in Cos-7 cells, the control antigen was either thepellet or supernatant of Cos-7 cells transfected with control emptyvector. Results are shown in Table 7. Reactivity with each of the newlydiscovered antigens was documented in at least one study subject.Overall, reactivity with U_(L) 49 was observed more frequently andsimilar to that for the known antigens gB2 and gD2. These data providesupport that human individuals, in addition to the index subjects inwhom the T-cell reactivity was originally described, are capable ofreacting to these antigenic HSV-derived proteins. TABLE 7 Antigenicityof known and of newly discovered HSV-2 antigens among a group of sevenrandomly chosen HSV-2 infected immunocompetent adults. ANTIGEN VP16 ofU_(L) 49 of HSV-2 gB2 gD2 HSV-2 HSV-2 n 7 5 5 0 5 % 100 71 71 0 71

Example 4 Detection of HSV-Specific CD8 CTL in Recurrent Genital HSV-2Lesions

[0153] This example demonstrates that specific CD8 CTL localize togenital HSV-2 lesions. This is shown by serial lesion biopsy studies ofrecurrent genital HSV-2 lesions using cells that have encounteredantigen/APC in situ and are not restimulated with antigen in vitro priorto readout assays.

[0154] To study the cDNA species derived from the positive genomic clonecontaining portions of ICP0 (Results), COS-7 cells (100 mm²) weretransfected with the ICP0 genomic clone, and total RNA was preparedafter 48 h. The primer used for cDNA synthesis(TGCTCTAGAGACTCGATCCCTGCGCGTCGG; XbaI site underlined) (SEQ ID NO: 7)was from the 3′-end of the HSV-2 DNA in the ICP0 genomic clone. Moloneymurine leukemia virus reverse transcriptase (Life Technologies) was usedper the manufacturer. To examine splicing, PCR used pfu cDNA polymerase,the above 3′-primer, and 5′-primer TAAGGTACCTGAACCCCGGCCCGGCACGAGC (SEQID NO: 8) (KpnI site). To isolate exon 1 (Dolan, A. et al., 1998, J.Virol. 72:2010) of ICP0, PCR used the same 5′-primer and 3′-primerTGCTCTAGACCAGGCGTGCGGGGCGGCGGG (SEQ ID NO: 9) (XbaI site). Reactionconditions were individually optimized. Product was digested with Acc65Iand XbaI, gel purified, and ligated into similarly treated pCDNA 3.1-His-B, and in-frame insertion was confirmed by sequencing.

[0155]

[0156] Full-length U_(L) 47 of HSV-2 was cloned by PCR into pCDNA3.1/His-C using 5′-primer CTAGGATCCCCTCCGGCCACCATGTCC (SEQ ID NO: 10) and3′-primer CGATCTAGACCTATGGGCGTGGCGGGC (SEQ ID NO: 11) (BamHI and XbaIsites underlined). Full-length U_(L) 46 of HSV-2 was cloned by PCR intopcDNA3.1/His-C with 5′-primer CGAGGATCCGTCTCCGCCATGCAACGCCG (SEQ ID NO:12) and 3′-primer CGCTCTAGATTTTAATGGCTCTGGTGTCG (SEQ ID NO: 13) (BamHIand XbaI sites underlined). Similarly, a construct expressing aa 1-590of U_(L) 47 was made by PCR, using the above 5′-primer, an appropriate3′-primer, and pCDNA3.1/His-C. Expression of aa 1-535 and 536-696 ofU_(L) 47 was driven by constructs derived from full-length U_(L) 47using a naturally occurring NotI site at aa 535. In-frame vector-HSV-2fusion at the 5′-end of the HSV-2 DNA was confirmed by sequencing ineach case.

[0157] Results TABLE 8 CTL activity and HLA restriction of CD8 clones,and initial results of expression cloning¹ CD8 CTL Clone 1874.1991.225101.1999.23 5491.2000.48 Autologous targets Mock 1.2 6 1.2 HSV-1 0.1 02.3 HSV-2 38.3 56.6 63.2 HSV-2 hr259 (ICP4⁻) 21.3 41.0 NDHSV-2/actinomycin D 45.1 35.8 49.2 HLA-mismatched targets Mock 0 2.5 9.8HSV-2 0 2.1 7.0 HLA restriction testing Matched allele A*0201 A*0201B*0702 Mock 3.3 0 5.1 HSV-2 65.2 33.4 69.1 Specificity Positive genomicclone C:1:F1:C7 C:2:C10:B9 UL49-pEGFP-C1 Nucleotides 102,875-101,383102,943-102,876 107,149-106,247 Predicted HSV-2 ORF(s) U_(L) 47 300-696. . . U_(L) 47 278-298 U_(L) 49 1-300 U_(L) 46 1-71 # clones are listedby indicating the positive pCDNA3.1/His A, B, or C library: positivelibrary plate: positive library well:positive final well. For5491.2000.48, full-length U_(L) 49 of HSV-2 in pEGFP-C1 (Clontech) waspositive. The nucleotide numbers and predicted amino acid numbers withinthe antigenic HSV-2 DNA fragments are given as reported for the HSV-2strain HG52 genomic sequence (28).

[0158] Recognition of Tegument HSV-2 Ags by CD 8 Cells

[0159] CD8 clone 5101.1999.23 recognized COS-7 cells co-transfected withHLA A*0201 and a HSV-2 Sau3aI fragment from bp 102,943-102,876 (Dolan,A. et al., 1998,J. Virol. 72:2010) (Table 8). The predicted fusionprotein contains HSV-2 U_(L) 47 aa 278-298. Reactivity with U_(L) 47 wasconfirmed by cotransfection of A*0201 and full-length HSV-2 U_(L) 47(Table 9). TABLE 9 Confirmation and localization of epitopes recognizedby CD8⁺ clones¹ T Cell Clone HSV-2 ORF and Predicted Amino Acids HLAcDNA IFN-γ(pg/ml) 5101.1999.23 None None 0 U_(L) 47 aa 1-696 (fulllength) None 0 None A*0201 0 U_(L) 47 aa 1-696 A*0201 >3000 1874.1991.22None None 0 U_(L) aa 1-696 None 0 None A*0201 0 U_(L) 46 aa 1-722 (fulllength) A*0201 0 U_(L) 47 aa 1-696 A*0201  2984 U_(L) 47 aa 1-535 A*02010 U_(L) 47 aa 1-590 A*0201 >3000 U_(L) 47 aa 536-696 A*0201 >30001874.1997.51 Genomic, nucleotides 1858-3022 None 0 None B*4501 0Genomic, nucleotides 1858-3022 B*4501 >600  ICP0 exon 1 cDNA aa 1-25B*4501 3.2 ICP0 exon 1/start of exon 2 cDNA aa 1-105 B*4501 >600  #1874.1997.51 is localized to aa 26-105 of ICP0. Values are mean ofduplicate IFN-γ secretion into the medium as measured by ELISA. ORF,Open reading frame.

[0160] The CD8 T cell clone 1874.1991.22 recognized COS-7 cellscotransfected with HLA A*0201 and a HSV-2 Sau3aI fragment from bp102,875-101,383 (Table 8). This fragment was predicted to contain theDNA encoding U_(L)47 aa 300-696, intervening DNA, and then aa 1-71 ofU_(L)46. Analysis of the 5′-vector-insert junction in C:2:C10:B9revealed out-of-frame translation of the initial U_(L)47 DNA. The insertis expected to contain the U_(L)46 promoter. The epitope, therefore,could be encoded by U_(L)46. C:2:C10:B9 also contains potential sites ofinternal initiation of translation within U_(L)47. U_(L)46 and U_(L)47were assayed separately in the COS-7 cotransfection assay (Table 9).U_(L)47 was active, whereas U_(L)46 was not. Truncation analysislocalized the epitope to aa 535-590 (Table 9).

[0161] The specificity of CD8 clone 5491.2000.48 was determined with apanel of partial- and full-length HSV-2 genes. The HSV-2 genes studiedwere previously shown to be recognized by CD4 T cell clones (See U.S.Pat. No. 6,375,952, issued Apr. 23, 2002). Only HSV-2 U_(L)49, whencotransfected with HLA B*0702, stimulated IFN-γ release by clone5491.2000.48 (FIG. 5).

[0162] HSV-2 gene U_(L)47 encodes protein VP13/14, whereas U_(L)49encodes VP22; both tegument proteins are loaded into the cytoplasm onvirion binding and entry. The small genomic HSV-2 fragment of U_(L)47recognized by clone 5101.1999.23 was scanned for peptides fitting theA*0201 binding motif (http://134.2.96.221/ andhttp://bimas.dcrt.nih.gov/molbio/hla_bind/). Peptide U_(L)47 (HSV-2)289-298 had a 50% effective concentration (EC₅₀) in the 1-10 nM range incytolysis assays (FIG. 6). U_(L)47 535-590 (Table 9) was similarlyanalyzed. Peptide 551-559 was active at 1 nM (FIG. 6). Potential HLAB*0702-binding peptides in U_(L)49 of HSV-2 were synthesized, and two(aa 47-55 and 14-22) were active at 1 μM. Titration (FIG. 6) showed thatU_(L)49 49-55 was highly active, with an EC₅₀ of <10 nM, whereas U_(L)4914-22 had activity only at 1 μM. The antigenic peptides in U_(L)47 andU_(L)49 contain significant amino acid sequence differences from thecorresponding predicted HSV-1 peptides (Dolan, A. et al., 1998,J. Virol.72:2010; McGeoch, D. J. et al., 1988,J. Gen. Virol. 69:1531), explainingtype-specific recognition of HSV-2 (Table 8).

[0163] Recognition of Immediate Early HSV-2 Protein ICP0 by CD8 T Cells

[0164] For clone 1874.1997.51, positive reactions to plasmid pools werepresent in each library. The active plasmids in each library contained agenomic Sau3AI fragment from nucleotides 1858-3022 (Dolan, A. et al.,1998, J. Virol. 72:2010). Nucleotide 2007 listed as T in the publishedsequence was read as C. In addition to 445 bp of 5′-untranslatedsequence, all of predicted exon 1, intron 1, and the first 234 bp ofpredicted exon 2 of ICP0 were present, preliminarily identifying theantigen as ICP0. Because alternative splicing of HSV-1 ICP0 has beendocumented at both the RNA and protein levels (Weber, P. C. et al.,1999, Virology 253:288; Carter, K. L., B. Roizman, 1996, Proc. Natl.Acad. Sci. USA 93:12535), the Ag-encoding mRNA species was firstidentified in COS-7 cells to determine how the ICP0 genomic clone wasspliced in this system. COS-7 cells were transfected with genomic cloneC:1:H3:B8 (Table 8), and cDNA was synthesized from total cellular RNAfollowed by PCR designed to amplify the spliced transcript. The size ofthe PCR product (˜300 bp) was consistent with the splicing out ofintron 1. Sequencing showed a slight difference from the reported splicepoint for mature HSV-2 ICP0 mRNA. Three base pairs encoding aa Q26 weremissing. Q26 was retained for peptide numbering (below). To determinewhether the antigenic peptide lay within exon 1 or exon 2, PCR wasrepeated with specific primers. The exon 1-partial exon 2 cDNA, but notexon 1 cDNA, was stimulatory for T cell clone 1874.1997.51 (fable 9),localizing the epitope to aa 26-105 in exon 2. Reactivity was confirmedin CTL assays using a recombinant vaccinia virus expressing ICP0. At E:T20:1, lysis of vaccinia ICP0-infected target cells was 52.1% comparedwith 2.3% for vaccinia wild type.

[0165] Two reported HLA B45-restricted epitopes, AEEAAGIGIL (SEQ ID NO:14) and GAETYVDGA (SEQ ID NO: 15), share with the B44 supertype apreference for negatively charged and hydrophobic amino acid side chainsat the P2 and P9 anchor positions. ICP0 (HSV-2) 92-105, containing thismotif, was active at 1 μM. Truncation yielded ICP0 (HSV-2) 92-101, withan EC₅₀ in the 1 nM range (FIG. 6).

[0166] Recognition of Skin-Derived Fibroblasts and Keratinocytes by CD8CTL Clones

[0167] Within lesions, HSV-2 is mainly present in keratinocytes. How MOI(amount of virus), time of infection, and pretreatment with IFN-γinfluenced lysis of dermal fibroblasts and keratinocytes wasinvestigated. For fibroblasts (FIG. 7), in the absence of IFN-γpretreatment, infection for 2 h led to detectable lysis, which increasedwith increasing MOI. Lysis was undetectable (<5% specific release at E:Tof 20:1) after overnight infection with MOI 1, 5, or 25. With IFN-γpretreatment, lysis was generally increased, but 2-h infection was stillsuperior. HLA-mismatched target cells were not lysed, even after peptideloading.

[0168] Keratinocytes showed some similarities and differences fromfibroblast as target cells (FIG. 7). IFN-γ pretreatment generallyincreased recognition, without leading to lysis of control cells. Incontrast to fibroblasts, 18-h infection was generally required. Weakcytolysis of cells infected for 2 h was noted only for IFN-γ-pretreatedtargets. Chromium release again correlated directly with the amount ofinfectious virus added, because no specific lysis was noted at MOI 1 or5.

[0169] TAP Dependence of Ag Processing for Recognition by HS V-2Tegument Protein Epitopes by CD 8 CTL

[0170] For each of the three CD8 clones studied, lysis of TAP-deficientcells after HSV-2 infection was greatly reduced in comparison towild-type EBV-LCL (Table 10). Greater than 90% of each of theTAP-deficient cell lines, as well as control wild-type LCL, werepermissive for viral infection and protein synthesis as evaluated byflow cytometry using mAb specific for envelope glycoprotein gD. Peptideloading was able to sensitize the TAP-deficient cells, confirming HLAclass I expression. TABLE 10 TAP dependence of processing of HSV-2tegument for presentation to CD8 T cells¹ Target Cells Mock PeptideHSV-2 CD8 clone 1874.1991.22 Controls 1874 2.5 54.8 30.7 5491 2.0 −2.5−1.2 TAP(−) 721.174 −3.9 90.0 1.3 T2 0.7 94.7 2.2 CD8 clone 5101.1999.23Controls 1874 0.8 52.5 18.2 5491 1.7 −2.1 −1.2 TAP(−) 721.174 0 31.7 2.9T2 −0.7 71.0 10.8 CD8 clone 5491.2000.48 Controls 1874 0.8 −2.7 13.65491 0.2 68.3 1.2 TAP(−) T2/B7.63 −0.4 57.8 3.4 T2 0.2 0.4 3.6 # (MOI10, 18 h). In contrast, peptide loading, but not HSV-2-infection, wasable to sensitize TAP-deficient cell lines. Similar data are shown forthe third clone, a HLA B*0702-restricted, U_(L) 49-specific CTL cloneand peptide U_(L) 49 49-57, using the B*0702 autologous EBV-LCL, 5491,the non-B*0702 EBV-LCL, 1874, and the TAP-deficient, HLAB*0702-containing # transfectant T2/B7.63. As an additional control, T2cells, which do not express B*0702, were not lysed after peptideloading.

[0171] Discussion

[0172] HSV-2 causes considerable morbidity and mortality, especially inneonates. Because of the chronic nature of the infection, thelimitations of antiviral therapy, and the frequency with whichtransmission is caused by asymptomatic shedding of the virus,vaccination is likely to be required to reduce new HSV-2 infections. Therecent report that vaccination with a specific adjuvant and an envelopeglycoprotein induced partial protection in HSV-1/HSV-2-seronegativewomen highlights both the potential efficacy of vaccination and the needfor improved formulations and markets of effective immunity.

[0173] Little is known about the specificity of human HSV-2-specific CD8CTL. The two published epitopes are type-common peptides withinglycoproteins B and D. At the nonclonal level, experiments usingrestimulation of PBMC, drug blocks, and vaccinia recombinants show thatHSV-1 ICP4, ICP27, ICP0, all immediate early proteins, HSV-1 earlyprotein ICP6, and possibly other true early proteins may be targets ofhuman CTL. HSV-1 early protein thymidine kinase (tk) is recognized byCD8 clones from PBMC of subjects treated with tk-transfected autologouscells, but this is likely a primary immune response. A PBMC-derived CD8T cell clone specific for a melanoma-associated protein (Melan A/MART-1)also reacted with a peptide from HSV-1 glycoprotein C.

[0174] U_(L)49 encodes VP22, a tegument protein required for viralreplication. U_(L)49 protein is also abundant in virions and deliveredinto the cytoplasm by virus entry. Lysis of EBV-LCL by tegument-specificCD8 CTL was not inhibited by blockade of gene transcription or infectionwith a replication-incompetent virus, consistent with the processing andpresentation of preformed virion input protein.

[0175] TAP-independent processing has been reported in other viralsystems. The examination of three discrete epitopes in tegument proteinsdid not reveal evidence for TAP-independent Ag processing of HSVepitopes. The CD8 response seems to “evade the evasion,” at least in thecases examined to date, while continuing to rely on TAP for Agprocessing.

[0176] Most studies of clonal CD8 responses have used EBV-LCL as targetcells. These cells are relatively resistant to HSV-mediated class Idown-regulation. For dermal fibroblasts, it was found that a short timeof infection (2 h) was adequate for target cell sensitization for lysisby tegument protein-specific CTL. Because the U_(L)47 and U_(L)49tegument proteins are synthesized with “late” kinetics, typicallystarting after 6 h or more of viral infection, these data are alsoconsistent with recognition of preformed Ag in fibroblasts. Lysis wasMOI dependent. Because HSV preparations typically contain a large numberof defective particles, it is likely that tegument proteins were alsobeing delivered into fibroblasts by noninfectious particles. After 18 hof infection, the fibroblasts were not lysed, regardless of MOI, similarto previous results with CD8 CTL clones of unknown fine specificity.IFN-y pretreatment was able to partially restore lysis of 18-h-infectedcells. In contrast to fibroblasts, recognition of keratinocytes after 18h of infection was superior to recognition after 2 h of infection. Thereason for the difference between fibroblasts and keratinocytes isunknown. IFN-γ pretreatment was able to restore some lysis of2-h-infected cells, and further improved recognition of 18-h-infectedcells.

[0177] Tegument proteins have not previously been described as targetsof the HSV-specific CD8 T cell response. CD4 responses to HSV-1 U_(L)47have been detected in HSV-mediated acute retinal necrosis. CD4 responsesto U_(L)49 are commonly detected among lesion-infiltratingHSV-2-specific clones. Because responses to U_(L)49 are also present inthe cornea in herpes stromal keratitis in humans, a disease that may bedriven by pathogenic Th1-like T cells, caution is warranted in usingthis protein as a vaccine. Overall, U_(L)49 is the only known HSV-2protein recognized by both CD4 and CD8 T cell clones recovered fromherpetic lesions. A unique intercellular transport pathway allows highlyefficient uptake of soluble U_(L)49 protein into a variety of epithelialcell types which could also intersect antigen processing pathways.

[0178] In summary, reactivity of lesion-infiltrating, HSV-2type-specific CD8 T cell clones with the tegument proteins encoded bygenes U_(L)47 and U_(L)49 (VP13/14 and VP22, respectively), and ICP0,are described for the first time. The data are consistent with amodulatory effect of ICP47 and/or vhs on the CD8 response to HSV. TAPfunction, but not viral gene transcription, is required for recognitionby U_(L)47- and U_(L)49-specific clones, consistent with processing ofpreformed virion input protein. Tegument-specific CD8 clones were ableto recognize skin-derived fibroblasts and keratinocytes. Responses werealso detectable in the PBMC of additional subjects.

Example 5 HSV-2 15-Mer Peptide Screening with CD8+ T Cells

[0179] HSV-2 seropositive donors were obtained (AD104, AD116, AD120,D477, HV5101, JH6376, EB5491, TM10062). Donors HV5101 and EB5491experience frequent anogenital lesion recurrences. Donors JH6376 andTM10062 experience few or no anogenital lesion recurrences.Leukopheresis PBMC were obtained from each donor after informed consent.Donor PBMC were HLA-typed by low resolution DNA typing methodology.Synthetic peptides (15 amino-acids in length and overlapping in sequenceby 11 amino-acids) were synthesized that spanned the following HSV-2polypeptides: U_(L)47 (aa 1-696), U_(L)49 (aa 1-300), ICP27 (aa 1-512).Peptides (5 mg each) were delivered in lyophilized form in glass vialsand dissolved at a concentration of 10 mg/ml in DMSO, transferred tosterile cryovials and stored at −20 degrees C. The peptides werescreened with CD8+ T cells purified from adherent macrophage-depletedPBMC. CD8+ T cells were purified by depletion of non-CD8+ cells using acommercial MACS bead kit (Miltenyi). CD8+ T cells purified in thismanner were generally >95% CD8+CD3+CD4-, as measured by flow cytometry(FACS). Peptides were screened by 24-hour co-culture of CD8+ T cells(2×10e5/well) and autologous dendritic cells (1×10e4/well) and peptides(10 μg/ml each) in 96-well ELISPOT plates that had been pre-coated withanti-human IFN-g antibody 1D1K (mAbTech). Peptides were initiallyscreened as pools of >/=10 peptides. ELISPOT plates were subsequentlydeveloped per a standard protocol. The number of spots per well wascounted using an automated video-microscopy ELISPOT reader. Peptides inpools scoring positive were subsequently tested individually in a secondELISPOT assay. For AD116, the novel peptides U_(L)49/21-35 (#6)andU_(L)49/193-208 (#49) scored positive both pooled and individually.AD116 also recognized the previously described B*0702-restricted epitopeU_(L)49/49-57 contained in 15-mer peptides U_(L)49/45-59 (#12) andU_(L)49/49-63 (#13). D477, HV5101, and JH6376 T cells recognized thepreviously described HLA-A*0201-restricted epitopes U_(L)47/289-297 andU_(L)47/550-559 contained in 15-mers #73/#74 and #137/#138,respectively. EB5491 T cells recognized the previously describedB*0702-restricted epitope U_(L)49/49-57 epitope contained in 15-merpeptides U_(L)49/45-59 (#12) and U_(L)49/49-63 (#13). D477 scorepositive for peptide pool U_(L)49 (#11-20). TM10062 did not scorepositive on any peptide pool from U_(L)47 or U_(L)49. Donor HLA TypesDonor: HLA-A HLA-B HLA-C AD104 24, 33 46, 58 01, 0302 AD116 0206, 240702, 35 0702, 1203 AD120 0211, 3303 1505, 4403 0303, 0706 D477 0201,2501 1501, 5101 0304, 12 HV5101 0101, 0201 0801, 57 06, 0701 JH63760201, 03 07, 44 05, 07 EB5491 01, 26 07, 08 07 TM10062 0201, 26 14, 2701, 08

[0180] !CD8+ T cell peptide-screening hits AD104 US3 #33 HSV-2AIDYVHCKGIIHRDI (SEQ ID NO: 16) HSV-1 AVDYIHRQGIIHRDI (SEQ ID NO: 17)AD116 UL47 #86 HSV-2 AVPLLSAGGAAPPHP (SEQ ID NO: 18) HSV-1AVPLLSAGGLVSPQS (SEQ ID NO: 19) UL49 #6 HSV-2 ELYYGPVSP-ADPESP (SEQ IDNO: 20) HSV-1 DLYYTPSSGMASPDSP (SEQ ID NO: 21) UL49 #12 HSV-2PMRARPRGEVRFLHY (SEQ ID NO: 22) HSV-1 QRSARQRGEVRFVQY (SEQ ID NO: 23)UL49 #13 HSV-2 RPRGEVRFLHYDEAG (SEQ ID NO: 24) HSV-1 RQRGEVRFVQYPESD(SEQ ID NO: 25) UL49 #49 HSV-2 VAGFNKRVFCAAVGR (SEQ ID NO: 26) HSV-1VAGFNKRVFCAAVGR (SEQ ID NO: 27) HV5101 UL47 #143 HSV-2 STAPEVGTYTPLRYAC(SEQ ID NO: 28) HSV-1 FTAPEVGTYTPLRYAC (SEQ ID NO: 29)

[0181]FIG. 8 shows the results for AD116. FIG. 9 shows the results forEB5491, TM10062 and HV5101. The peptide hit indicated with a “1”represents U_(L)49, amino acids 49-57, B*0702-restricted. FIG. 10 showsresults for AD104, AD116, AD120 and D477. The peptide hit indicated witha “1” represents peptide #54. The peptide hit indicated with a “2”represents peptide #49. The peptide hit indicated with a “3” representsU_(L)49, amino acids 49-57, B*0702-restricted. The peptide hit indicatedwith a “4” represents peptide #6.

Example 6 Detection of HSV-Specific T-Cell Responses in CervicalLymphocytes

[0182] Mucosal immune responses are segregated from PBMC, andlocalization of HSV-specific CTL to the mucosa of mice is associatedwith protection from vaginal inoculation. This example demonstrates thatHSV-specific T cells, including CD8+ cells, can be detected in cervicallymphocytes.

[0183] Cells from a representative cervical cytobrush specimen werecollected during an active genital HSV-2 outbreak and expanded in bulkwith PHA/IL-2, and subsequently analyzed for HSV-specific proliferativeand cytotoxic responses. Proliferation and cytotoxicity assays usedautologous PBMC or LCL as APC as described above for skin-derivedlymphocytes. Anti-HLA class I mAb W6/32 or anti-HLA DR mAb L243 wereused as described (Koelle D M et al., J. Virol. 1994, 68:2803-10; KoelleD M et al., J. Infect. Dis. 1994, 169:956-61). Antigen-specificproliferative responses and cytotoxic responses were present.Fractionation and mAb inhibition studies show a contribution of CD8 CTLto the cytotoxic response.

Example 7 Detection of HSV-Specific T-Cell Responses in Primary GenitalHSV-2 Lesions

[0184] In this example, biopsy specimens were collected from a patientpresenting with symptoms consistent with primary genital HSV-2infection. The phenotypes of the collected cells were determined, andLIL and PBMC from the specimens were subjected to proliferative andcytotoxicity assays. The results show that the HSV-specificproliferative and cytotoxic responses of CTL present in primary genitalHSV-2 lesions are typical of those detected during recurrent disease.

[0185] CW7477 developed dysuria, fever, buttock, and lower abdomenlesions three days after his last sexual contact. Lesions lasted 13 daysand grew HSV-2. Acyclovir treatment was begun on day four of symptoms.Biopsies were done on days four and seven. Serostatus was atypicalpositive (only a few bands present on immunoblot) at day four, with morebands, but still less than most convalescent sera, on day 26, byenhanced chemiluminescence (ECL; Dalessio J. and Ashley R., J. Clin.Microbiol. 1992, 30(4):1005-7) variant of type-specific HSV-2immunoblot. The clinical and laboratory data were consistent withprimary genital HSV-2 infection. Biopsy specimens were obtained on dayfour and seven of symptoms and bulk LIL expanded with PHA/IL-2 asdescribed above.

[0186] The phenotype of the expanded cells was split between CD4 and CD8cells, with 15-25% CD3+/CD16,56+ cells and 5-10% TCR γδ+cells in theLIL. In comparison, cells from normal skin had almost no CD16,56 (+)events and no TCR γδ cells. The nature of the CD3+/CD16,56+ cells isunknown but these are frequently seen in expanded LIL. The antibodycocktail has a combination of αCD16-PE and αCD56-PE.

[0187] The HSV-specific proliferative and cytotoxic responses werefairly typical of those detected during recurrent disease (Koelle D M etal., J. Clin. Invest. 1998; 101:1500-1508). Cross-reactive responses toHSV-1 and HSV-2 were present, as were antigen-specific responses to HSVglycoproteins. Normal skin responses were low, and PBMC responses weredeveloping by day 15.

Example 8 Identification of an ICP0 Antigen Recognized by HSV-SpecificCD8 CTL

[0188] This example demonstrates, via expression cloning, theantigenicity of ICP0. In particular, an epitope within amino acids92-101 of ICP0 is identified. In addition, the antigenicity of ICP0 isconfirmed using vaccinia. The amino acid numbering uses the nomenclatureand numbering of Dolan et al., J. Virol 1998, 72:2010-21. The methodsused herein are described in U.S. Pat. No. 6,413,518, issued Jul. 2,2002.

[0189] Results

[0190] All HSV-specific CD8 clones released IFN-γ in a specific manner.In addition, the utility of the interferon-gamma assay was examined as aconfirmatory test for HLA restriction. Clone RW51 specifically releasedinterferon-gamma after exposure to Cos-7 cells transfected with HLAB*4501, but not with A*0201, and infection with HSV-2. TABLE 12Secretion of interferon-gamma of CD8 TCC RW51 in response to Cos-7 cellstransfected with various DNAs (or peptide loaded at 1 μM) measured byELISA in pg/ml. Responses of 5 × 10⁴ TCC to 7 × 10³ Cos-7 cells checkedat 24 hours. HSV-2 DNA or peptide HLA class empty pool clone ICP0 ICP0ICP0 I cDNA vector A1:H3 A1:H3:B8 exon 1 exon 1,2 92-105 empty not not<2  <2 <2  <2 vector done done B*4501 <2 420 >600 <2 >600 1,100

[0191] To choose peptides efficiently, a HLA B45 binding motif wasderived from B45-restricted peptides, and pool sequence from peptideseluted from B*4501. The motif is glutamic acid at position 2,hydrophobic at position 10 (P1 and P9 in “binding” nomenclature(Rammansee H -G, Current Opinion in Immunology 1995, 7:85-96)). PeptideICP0 92-105 (AERQGSPTPADAQG; SEQ ID NO: 30) was active in CTL (FIG. 14)and interferon-gamma (Table 12) assays. Other candidate exon 2 peptideswere not. The high EC₅₀ value (˜1 μM) may be due to the carboxy-terminustail predicted to lie outside the peptide-binding groove and reducebinding to HLA B*4501. Vaccinia-ICP0 from B. Rouse (Manickan E et al.,J. Virol. 1995, 69:4711-16) was grown and titered (Koelle D M et al., J.Virol. 1994, 68:2803-10). Clone RW51 specifically lysed vac-ICP0 targets(FIG. 12). The availability of the vaccinia was fortuitous, and notrequired to confirm the result of expression cloning. To narrow down theepitope, a peptide comprising amino acids 92-101 of ICP0 (AERQGSPTTP;SEQ ID NO: 31) was synthesized. The IC₅₀ for this peptide is between 1and 10 nanomolar (FIG. 13).

[0192] To confirm that patients with HSV-2 infection have T-cellsreactive with the newly discovered T-cell antigen circulating in theirperipheral blood, peripheral blood mononuclear cells (PBMC) from thepatient from whom the lesion-derived clone RW51 was recovered werepeptide stimulated. PBMC were cultured for three days at 2×10⁶ cells per1.88 cm² well in 2 ml of T-cell medium containing 1.0 μg/ml peptideHSV-2 ICP0 92-101. On the fourth day, IL-2 (32 units/ml) was added. Onthe eighth day, the cells were washed and restimulated in the same sizewell with an additional 2×10⁶ autologous, irradiated (3300 rad gammairradiation) PBMC, 1.0 μg/ml of the same peptide, and IL-2 (32 U/ml).

[0193] Responders were cultured for an additional nine days in thepresence of IL-2 and expanded as necessary. Cytotoxicity assay wasperformed using autologous or HLA class I-mismatched LCL treated eitherwith nothing, peptide HSV-2 ICP0 92-101 at 1 μg/ml for 18 hours, orinfection with HSV-2 strain 333 at MOI 10 for 18 hours. The cytotoxicityassay was a standard four-hour ⁵¹Cr release assay.

[0194] The results (FIG. 14) show that stimulation of PBMC with peptideHSV-2 ICP0 92-101 was able to stimulate cells with cytotoxicity towardsHSV-2 infected cells, and that this activity was not present against HLAclass I-mismatched cells. For comparison, the index T-cell clone RW51was also used as an effector cell in this assay and displayedcomparable, although slightly higher, cytotoxicity at the effector totarget ratio of 10:1 shown in FIG. 14.

Example 9 Identification of Additional ICP0 Antigens Recognized byHSV-Specific CTL

[0195] This example demonstrates, via use of a different population ofCD8+ T cells from a different human subject, the specific recognition ofamino acids 743-751 by lesion-derived T cells. The recognition eventinvolves HLA allele B*0702, which is relatively common (approximately10%) in the human population. In addition, amino acids 288-307 of ICP0have been found to be specifically recognized by lesion-derived T cells.

Example 10 ICP0 Stimulation of CTL Responses in Additional HLA-B45Subjects

[0196] This example demonstrates that other HLA-B45 positive donors havedetectable CD8+ T cell responses to the previously defined ICP0 92-101peptide.

[0197] Peptide restimulation in bulk format are appropriate forsensitive detection of CTL, while lesion derived antigen (LDA) formatsyield CTL levels, but require prolonged cell replication for detection.In this example, 4×10⁶ PBMC in 2 ml T-cell medium were stimulated with1μμg/ml HSV-2 peptides, and IL-2 (10-30 U/ml) was added on day 3. On day8, responders were washed and restimulated in 2 ml with 2×10⁶ autologousirradiated PBMC, fresh peptide, and IL-2, and split as necessary untilassay on day 14-16. For two HLA B*4501-bearing persons including theindex subject, convincing HLA class-restricted CD8 CTL were detectedthat not only lysed peptide-loaded targets, but also killedHSV-2-infected targets and were inhibited by anti-class I mAb (Table13). TABLE 13 Lysis of HLA B*4501 LCL by PBMC stimulated with peptideHSV-2 ICP0 92-101, or (+) control clone RW.1997.51. Results are percentspecific release in four-hour CTL assays at effector to target ratio of10:1-20:1. target¹ RW RW RW RW HSV-2/ HV HV HV effector mock peptide¹HSV-2 anti-class I² mock peptide HSV-2 RW PBMC 1 45.3 48.2 12.2 0 −1 0PO PBMC 0 54.9 33.5 5.8 4 −1 0 clone 0 65.3 67.3 5.2 1 0 2 RW. 1997.51

Example 11 Definition of Additional T-Cell Epitopes in Tegument ProteinU_(L)48 (VP16)

[0198] Three epitopes within VP16, all HSV-2 type-specific werepreviously identified (K. R. Jerome et al., 1998, J. Virol.,72:436-441), and proliferative responses to full-length VP16 in bulkcultures of genital HSV-2 lesion-infiltrating lymphocytes from four ofseven (57%) patients were detected (D. M. Koelle et al., 1998,J. Clin.Invest., 101:1500-09). Additional peptide epitopes were sought withinVP16 by two strategies. The first strategy involved screening panels ofclones recovered from lesion vesicle fluid for reactivity withrecombinant VP16 of HSV-2 followed by epitope mapping with peptides.Peptides containing amino acids 185-197 and the overlapping pair 209-221and 213-225 were stimulatory for TCC RH.13 and KM.7, respectively. Allother VP16 peptides tested were negative (<500 cpm). The second strategyinvolved using PBMC as starting material and secondary in vitrostimulation with recombinant baculovirus-derived VP16. Clones (BM.17 andSB.17) from two individuals recognized the same peptide (amino acids437-449) as well as β-gal-VP16 fusion protein and whole virus. All threenewly defined VP16 epitopes were type-common, shared by HSV-1 and HSV-2whole virus preparations, as expected from sequence data (A. Cress andS. J. Triezenberg, 1991, Gene, 103:235-238). TABLE 14 Epitopes withinU_(L)48 (VP16) of HSV-2 recognized by lesion- and PBMC-derived CD4 TCC.Data are delta cpm [³H] thymidine incorporation compared to media, whichwas less than 500 cpm in each case. recombinant HSV-2 whole virusprotein¹ TCC antigen VP16 1- β-gal-VP16 HSV-2 VP16 peptide name originHSV-1 HSV-2 492 180-492 amino acids delta cpm newly reported epitopesRH.13 lesion  3,340  3,407 32,991 nd 185-197 55,614 KM.7 lesion  6,093 5,847  5,627 nd 209-221 10,075 BM.17 PBMC 30,784 13,777 nd 45,958437-449 79,723 SB.17 PBMC  2,207  4,187 nd 12,178 437-449 36,442previously reported epitopes ESL4.34 lesion 256  8,780 17,302 nd 389-40112,968 393-405 95,942 ESL3.334 lesion 253 14,232 22,754 16,434 430-44427,283 1A.B.25 lesion 414 33.493 24,919 41,123 431-440 38,664

[0199] TABLE 15 Cytolytic activity of lesion-derived, tegument-specificCD4 TCC with summary of fine specificity and HLA restriction. Resultsare percent specific release at an effector to target ratio of 20:1except ESL4.34 (10:1). Auto = autologous EBV-LCL as target cells; allo =allogeneic EBV-LCL mismatched at the relevant HLA locus (if known) ormismatch at HLA DR and DQ. cytolysis assay target HLA auto auto autoallo allo TCC specificity¹ restriction² HSV-2 peptide mock HSV-2 peptideallo mock newly reported epitopes RH.13 VP16 185- DR⁴ 62.5 55.2 −0.9 9.60.3 1.8 197 KM.7 VP16 209- DR⁴ 38.7 43.6 2.7 −2.2 4.3 −1.1 221 BM.17VP16 437- DQB1*0501 10 1 28.5 −0.3 nd nd nd 449 SB.17 VP16 437-DQB1*0501 48.7 60.6 5.4 nd nd nd 449 previously described epitopesESL4.34 VP16 393- DRB1*0402  2.1 10.4 1.0 0.5 0.6 0.3 405 ESL3.334 VP16430- DQB1*0302 12.3 33.6 0.7 1.4 0.3 2.2 444 1A.B.25 VP16 431- DQB1*020124.3 42.2 1.9 1.7 2.1 −0.4 440

[0200] The HLA restriction of TCC BM.17 was studied in detail.Proliferation of TCC BM.17 and the similar clone SB.17 was inhibited 90%by anti-DQ, but less than 25% by anti-DR or anti-DP mAb. Donors BM andSB are heterozygous for HLA DQB1*0201/0501. At high 10 concentrations ofpeptide, both DQB1*0201- and DQB1*0501 homozygous EBV-LCL appeared topresent antigen to TCC BM.17. However, DQB1*0501 homozygous cellspresented peptide much more efficiently than DQB1*0201 homozygous cells(FIG. 19). Thus, three different but overlapping epitopes in VP16431-449 are presented by HLA DQB1*0302, DQB1*0201, and DQB1*0501.

[0201] CTL Activiy of Tegument-Specific CD4 T-Cell Clones

[0202] Cytotoxic activities of the CD4 TCC with newly and previouslyidentified specificities were tested using EBV-LCL target cells. Allclones tested displayed cytolytic activity towards peptide-loaded targetcells. Cytolytic activity against target cells infected with HSV-2showed some variability. Among the seven VP16-specific T-cell clonestested, six displayed greater than 10% cytotoxicity towardsHSV-2-infected target cells.

[0203] An additional epitope, included in amino acids 288-307 of U_(L)48(VP16) (RLRELNHIREHLNLPLVRSA; SEQ ID NO: 32), was demonstrated to havereactivity with a CD4+ T cell clone. This epitope is recognized inassociation with the HLA class II molecule DRBI*1501, which is fairlyprevalent in most human populations.

[0204] Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1 32 1 825 PRT Herpes simplex virus-2 (HSV-2) 1 Met Glu Pro Arg Pro GlyThr Ser Ser Arg Ala Asp Pro Gly Pro Glu 1 5 10 15 Arg Pro Pro Arg GlnThr Pro Gly Thr Gln Pro Ala Ala Pro His Ala 20 25 30 Trp Gly Met Leu AsnAsp Met Gln Trp Leu Ala Ser Ser Asp Ser Glu 35 40 45 Glu Glu Thr Glu ValGly Ile Ser Asp Asp Asp Leu His Arg Asp Ser 50 55 60 Thr Ser Glu Ala GlySer Thr Asp Thr Glu Met Phe Glu Ala Gly Leu 65 70 75 80 Met Asp Ala AlaThr Pro Pro Ala Arg Pro Pro Ala Glu Arg Gln Gly 85 90 95 Ser Pro Thr ProAla Asp Ala Gln Gly Ser Cys Gly Gly Gly Pro Val 100 105 110 Gly Glu GluGlu Ala Glu Ala Gly Gly Gly Gly Asp Val Cys Ala Val 115 120 125 Cys ThrAsp Glu Ile Ala Pro Pro Leu Arg Cys Gln Ser Phe Pro Cys 130 135 140 LeuHis Pro Phe Cys Ile Pro Cys Met Lys Thr Trp Ile Pro Leu Arg 145 150 155160 Asn Thr Cys Pro Leu Cys Asn Thr Pro Val Ala Tyr Leu Ile Val Gly 165170 175 Val Thr Ala Ser Gly Ser Phe Ser Thr Ile Pro Ile Val Asn Asp Pro180 185 190 Arg Thr Arg Val Glu Ala Glu Ala Ala Val Arg Ala Gly Thr AlaVal 195 200 205 Asp Phe Ile Trp Thr Gly Asn Pro Arg Thr Ala Pro Arg SerLeu Ser 210 215 220 Leu Gly Gly His Thr Val Arg Ala Leu Ser Pro Thr ProPro Trp Pro 225 230 235 240 Gly Thr Asp Asp Glu Asp Asp Asp Leu Ala AspVal Asp Tyr Val Pro 245 250 255 Pro Ala Pro Arg Arg Ala Pro Arg Arg GlyGly Gly Gly Ala Gly Ala 260 265 270 Thr Arg Gly Thr Ser Gln Pro Ala AlaThr Arg Pro Ala Pro Pro Gly 275 280 285 Ala Pro Arg Ser Ser Ser Ser GlyGly Ala Pro Leu Arg Ala Gly Val 290 295 300 Gly Ser Gly Ser Gly Gly GlyPro Ala Val Ala Ala Val Val Pro Arg 305 310 315 320 Val Ala Ser Leu ProPro Ala Ala Gly Gly Gly Arg Ala Gln Ala Arg 325 330 335 Arg Val Gly GluAsp Ala Ala Ala Ala Glu Gly Arg Thr Pro Pro Ala 340 345 350 Arg Gln ProArg Ala Ala Gln Glu Pro Pro Ile Val Ile Ser Asp Ser 355 360 365 Pro ProPro Ser Pro Arg Arg Pro Ala Gly Pro Gly Pro Leu Ser Phe 370 375 380 ValSer Ser Ser Ser Ala Gln Val Ser Ser Gly Pro Gly Gly Gly Gly 385 390 395400 Leu Pro Gln Ser Ser Gly Arg Ala Ala Arg Pro Arg Ala Ala Val Ala 405410 415 Pro Arg Val Arg Ser Pro Pro Arg Ala Ala Ala Ala Pro Val Val Ser420 425 430 Ala Ser Ala Asp Ala Ala Gly Pro Ala Pro Pro Ala Val Pro ValAsp 435 440 445 Ala His Arg Ala Pro Arg Ser Arg Met Thr Gln Ala Gln ThrAsp Thr 450 455 460 Gln Ala Gln Ser Leu Gly Arg Ala Gly Ala Thr Asp AlaArg Gly Ser 465 470 475 480 Gly Gly Pro Gly Ala Glu Gly Gly Pro Gly ValPro Arg Gly Thr Asn 485 490 495 Thr Pro Gly Ala Ala Pro His Ala Ala GluGly Ala Ala Ala Arg Pro 500 505 510 Arg Lys Arg Arg Gly Ser Asp Ser GlyPro Ala Ala Ser Ser Ser Ala 515 520 525 Ser Ser Ser Ala Ala Pro Arg SerPro Leu Ala Pro Gln Gly Val Gly 530 535 540 Ala Lys Arg Ala Ala Pro ArgArg Ala Pro Asp Ser Asp Ser Gly Asp 545 550 555 560 Arg Gly His Gly ProLeu Ala Pro Ala Ser Ala Gly Ala Ala Pro Pro 565 570 575 Ser Ala Ser ProSer Ser Gln Ala Ala Val Ala Ala Ala Ser Ser Ser 580 585 590 Ser Ala SerSer Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser 595 600 605 Ala SerSer Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser Ala 610 615 620 SerSer Ser Ala Gly Gly Ala Gly Gly Ser Val Ala Ser Ala Ser Gly 625 630 635640 Ala Gly Glu Arg Arg Glu Thr Ser Leu Gly Pro Arg Ala Ala Ala Pro 645650 655 Arg Gly Pro Arg Lys Cys Ala Arg Lys Thr Arg His Ala Glu Gly Gly660 665 670 Pro Glu Pro Gly Ala Arg Asp Pro Ala Pro Gly Leu Thr Arg TyrLeu 675 680 685 Pro Ile Ala Gly Val Ser Ser Val Val Ala Leu Ala Pro TyrVal Asn 690 695 700 Lys Thr Val Thr Gly Asp Cys Leu Pro Val Leu Asp MetGlu Thr Gly 705 710 715 720 His Ile Gly Ala Tyr Val Val Leu Val Asp GlnThr Gly Asn Val Ala 725 730 735 Asp Leu Leu Arg Ala Ala Ala Pro Ala TrpSer Arg Arg Thr Leu Leu 740 745 750 Pro Glu His Ala Arg Asn Cys Val ArgPro Pro Asp Tyr Pro Thr Pro 755 760 765 Pro Ala Ser Glu Trp Asn Ser LeuTrp Met Thr Pro Val Gly Asn Met 770 775 780 Leu Phe Asp Gln Gly Thr LeuVal Gly Ala Leu Asp Phe His Gly Leu 785 790 795 800 Arg Ser Arg His ProTrp Ser Arg Glu Gln Gly Ala Pro Ala Pro Ala 805 810 815 Gly Asp Ala ProAla Gly His Gly Glu 820 825 2 490 PRT Herpes simplex virus-2 (HSV-2) 2Met Asp Leu Leu Val Asp Asp Leu Phe Ala Asp Ala Asp Gly Val Ser 1 5 1015 Pro Pro Pro Pro Arg Pro Ala Gly Gly Pro Lys Asn Thr Pro Ala Ala 20 2530 Pro Pro Leu Tyr Ala Thr Gly Arg Leu Ser Gln Ala Gln Leu Met Pro 35 4045 Ser Pro Pro Met Pro Val Pro Pro Ala Ala Leu Phe Asn Arg Leu Leu 50 5560 Asp Asp Leu Gly Phe Ser Ala Gly Pro Ala Leu Cys Thr Met Leu Asp 65 7075 80 Thr Trp Asn Glu Asp Leu Phe Ser Gly Phe Pro Thr Asn Ala Asp Met 8590 95 Tyr Arg Glu Cys Lys Phe Leu Ser Thr Leu Pro Ser Asp Val Ile Asp100 105 110 Trp Gly Asp Ala His Val Pro Glu Arg Ser Pro Ile Asp Ile ArgAla 115 120 125 His Gly Asp Val Ala Phe Pro Thr Leu Pro Ala Thr Arg AspGlu Leu 130 135 140 Pro Ser Tyr Tyr Glu Ala Met Ala Gln Phe Phe Arg GlyGlu Leu Arg 145 150 155 160 Ala Arg Glu Glu Ser Tyr Arg Thr Val Leu AlaAsn Phe Cys Ser Ala 165 170 175 Leu Tyr Arg Tyr Leu Arg Ala Ser Val ArgGln Leu His Arg Gln Ala 180 185 190 His Met Arg Gly Arg Asn Arg Asp LeuArg Glu Met Leu Arg Thr Thr 195 200 205 Ile Ala Asp Arg Tyr Tyr Arg GluThr Ala Arg Leu Ala Arg Val Leu 210 215 220 Phe Leu His Leu Tyr Leu PheLeu Ser Arg Glu Ile Leu Trp Ala Ala 225 230 235 240 Tyr Ala Glu Gln MetMet Arg Pro Asp Leu Phe Asp Gly Leu Cys Cys 245 250 255 Asp Leu Glu SerTrp Arg Gln Leu Ala Cys Leu Phe Gln Pro Leu Met 260 265 270 Phe Ile AsnGly Ser Leu Thr Val Arg Gly Val Pro Val Glu Ala Arg 275 280 285 Arg LeuArg Glu Leu Asn His Ile Arg Glu His Leu Asn Leu Pro Leu 290 295 300 ValArg Ser Ala Ala Ala Glu Glu Pro Gly Ala Pro Leu Thr Thr Pro 305 310 315320 Pro Val Leu Gln Gly Asn Gln Ala Arg Ser Ser Gly Tyr Phe Met Leu 325330 335 Leu Ile Arg Ala Lys Leu Asp Ser Tyr Ser Ser Val Ala Thr Ser Glu340 345 350 Gly Glu Ser Val Met Arg Glu His Ala Tyr Ser Arg Gly Arg ThrArg 355 360 365 Asn Asn Tyr Gly Ser Thr Ile Glu Gly Leu Leu Asp Leu ProAsp Asp 370 375 380 Asp Asp Ala Pro Ala Glu Ala Gly Leu Val Ala Pro ArgMet Ser Phe 385 390 395 400 Leu Ser Ala Gly Gln Arg Pro Arg Arg Leu SerThr Thr Ala Pro Ile 405 410 415 Thr Asp Val Ser Leu Gly Asp Glu Leu ArgLeu Asp Gly Glu Glu Val 420 425 430 Asp Met Thr Pro Ala Asp Ala Leu AspAsp Phe Asp Leu Glu Met Leu 435 440 445 Gly Asp Val Glu Ser Pro Ser ProGly Met Thr His Asp Pro Val Ser 450 455 460 Tyr Gly Ala Leu Asp Val AspAsp Phe Glu Phe Glu Gln Met Phe Thr 465 470 475 480 Asp Ala Met Gly IleAsp Asp Phe Gly Gly 485 490 3 300 PRT Herpes simplex virus-2 (HSV-2) 3Met Thr Ser Arg Arg Ser Val Lys Ser Cys Pro Arg Glu Ala Pro Arg 1 5 1015 Gly Thr His Glu Glu Leu Tyr Tyr Gly Pro Val Ser Pro Ala Asp Pro 20 2530 Glu Ser Pro Arg Asp Asp Phe Arg Arg Gly Ala Gly Pro Met Arg Ala 35 4045 Arg Pro Arg Gly Glu Val Arg Phe Leu His Tyr Asp Glu Ala Gly Tyr 50 5560 Ala Leu Tyr Arg Asp Ser Ser Ser Asp Asp Asp Glu Ser Arg Asp Thr 65 7075 80 Ala Arg Pro Arg Arg Ser Ala Ser Val Ala Gly Ser His Gly Pro Gly 8590 95 Pro Ala Arg Ala Pro Pro Pro Pro Gly Gly Pro Val Gly Ala Gly Gly100 105 110 Arg Ser His Ala Pro Pro Ala Arg Thr Pro Lys Met Thr Arg GlyAla 115 120 125 Pro Lys Ala Ser Ala Thr Pro Ala Thr Asp Pro Ala Arg GlyArg Arg 130 135 140 Pro Ala Gln Ala Asp Ser Ala Val Leu Leu Asp Ala ProAla Pro Thr 145 150 155 160 Ala Ser Gly Arg Thr Lys Thr Pro Ala Gln GlyLeu Ala Lys Lys Leu 165 170 175 His Phe Ser Thr Ala Pro Pro Ser Pro ThrAla Pro Trp Thr Pro Arg 180 185 190 Val Ala Gly Phe Asn Lys Arg Val PheCys Ala Ala Val Gly Arg Leu 195 200 205 Ala Ala Thr His Ala Arg Leu AlaAla Val Gln Leu Trp Asp Met Ser 210 215 220 Arg Pro His Thr Asp Glu AspLeu Asn Glu Leu Leu Asp Leu Thr Thr 225 230 235 240 Ile Arg Val Thr ValCys Glu Gly Lys Asn Leu Leu Gln Arg Ala Asn 245 250 255 Glu Leu Val AsnPro Asp Ala Ala Gln Asp Val Asp Ala Thr Ala Ala 260 265 270 Ala Arg GlyArg Pro Ala Gly Arg Ala Ala Ala Thr Ala Arg Ala Pro 275 280 285 Ala ArgSer Ala Ser Arg Pro Arg Arg Pro Leu Glu 290 295 300 4 21 PRT Herpessimplex virus-2 (HSV-2) 4 Gly Gly Pro Val Gly Ala Gly Gly Arg Ser HisAla Pro Ala Arg Thr 1 5 10 15 Pro Lys Met Thr Arg 20 5 28 DNA ArtificialSequence primer 5 ggaagatcta cctctcgccg ctccgtca 28 6 29 DNA ArtificialSequence primer 6 ccggaattct tgtctgtcgt ctgaacgcg 29 7 30 DNA ArtificialSequence primer 7 tgctctagag actcgatccc tgcgcgtcgg 30 8 31 DNAArtificial Sequence primer 8 taaggtacct gaaccccggc ccggcacgag c 31 9 30DNA Artificial Sequence primer 9 tgctctagac caggcgtgcg gggcggcggg 30 1027 DNA Artificial Sequence primer 10 ctaggatccc ctccggccac catgtcc 27 1127 DNA Artificial Sequence primer 11 cgatctagac ctatgggcgt ggcgggc 27 1229 DNA Artificial Sequence primer 12 cgaggatccg tctccgccat gcaacgccg 2913 29 DNA Artificial Sequence primer 13 cgctctagat tttaatggct ctggtgtcg29 14 10 PRT Artificial Sequence epitope 14 Ala Glu Glu Ala Ala Gly IleGly Ile Leu 1 5 10 15 10 PRT Artificial Sequence epitope 15 Gly Ala GluThr Phe Tyr Val Asp Gly Ala 1 5 10 16 15 PRT Herpes simplex virus-2(HSV-2) 16 Ala Ile Asp Tyr Val His Cys Lys Gly Ile Ile His Arg Asp Ile 15 10 15 17 15 PRT Herpes simplex virus-1 (HSV-1) 17 Ala Val Asp Tyr IleHis Arg Gln Gly Ile Ile His Arg Asp Ile 1 5 10 15 18 15 PRT Herpessimplex virus-2 (HSV-2) 18 Ala Val Pro Leu Leu Ser Ala Gly Gly Ala AlaPro Pro His Pro 1 5 10 15 19 15 PRT Herpes simplex virus-1 (HSV-1) 19Ala Val Pro Leu Leu Ser Ala Gly Gly Leu Val Ser Pro Gln Ser 1 5 10 15 2015 PRT Herpes simplex virus-2 (HSV-2) 20 Glu Leu Tyr Tyr Gly Pro Val SerPro Ala Asp Pro Glu Ser Pro 1 5 10 15 21 16 PRT Herpes simplex virus-1(HSV-1) 21 Asp Leu Tyr Tyr Thr Pro Ser Ser Gly Met Ala Ser Pro Asp SerPro 1 5 10 15 22 15 PRT Herpes simplex virus-2 (HSV-2) 22 Pro Met ArgAla Arg Pro Arg Gly Glu Val Arg Phe Leu His Tyr 1 5 10 15 23 15 PRTHerpes simplex virus-1 (HSV-1) 23 Gln Arg Ser Ala Arg Gln Arg Gly GluVal Arg Phe Val Gln Tyr 1 5 10 15 24 15 PRT Herpes simplex virus-2(HSV-2) 24 Arg Pro Arg Gly Glu Val Arg Phe Leu His Tyr Asp Glu Ala Gly 15 10 15 25 15 PRT Herpes simplex virus-1 (HSV-1) 25 Arg Gln Arg Gly GluVal Arg Phe Val Gln Tyr Pro Glu Ser Asp 1 5 10 15 26 15 PRT Herpessimplex virus-2 (HSV-2) 26 Val Ala Gly Phe Asn Lys Arg Val Phe Cys AlaAla Val Gly Arg 1 5 10 15 27 15 PRT Herpes simplex virus-1 (HSV-1) 27Val Ala Gly Phe Asn Lys Arg Val Phe Cys Ala Ala Val Gly Arg 1 5 10 15 2816 PRT Herpes simplex virus-2 (HSV-2) 28 Ser Thr Ala Pro Glu Val Gly ThrTyr Thr Pro Leu Arg Tyr Ala Cys 1 5 10 15 29 16 PRT Herpes simplexvirus-1 (HSV-1) 29 Phe Thr Ala Pro Glu Val Gly Thr Tyr Thr Pro Leu ArgTyr Ala Cys 1 5 10 15 30 14 PRT Herpes simplex virus-2 (HSV-2) 30 AlaGlu Arg Gln Gly Ser Pro Thr Pro Ala Asp Ala Gln Gly 1 5 10 31 10 PRTHerpes simplex virus-2 (HSV-2) 31 Ala Glu Arg Gln Gly Ser Pro Thr ThrPro 1 5 10 32 20 PRT Herpes simplex virus-2 (HSV-2) 32 Arg Leu Arg GluLeu Asn His Ile Arg Glu His Leu Asn Leu Pro Leu 1 5 10 15 Val Arg SerAla 20

What is claimed is:
 1. A pharmaceutical composition comprising a herpessimplex virus (HSV) polypeptide, wherein the polypeptide comprises aU_(L)48, U_(L)49 or ICP0 protein, and a pharmaceutically acceptablecarrier.
 2. The pharmaceutical composition of claim 1, wherein theprotein comprises amino acids 185-197, 209-221, 288-307 or 430-449 ofU_(L)48 (SEQ ID NO: 2), amino acids 14-22, 21-35, 45-59, 49-57, 49-63,105-190, 177-220 or 193-208 of U_(L)49 (SEQ ID NO: 3), or amino acids92-101, 92-105, 288-307 or 743-751 of ICP0 (SEQ ID NO: 1).
 3. Thepharmaceutical composition of claim 1, wherein the polypeptide is afusion protein.
 4. The pharmaceutical composition of claim 3, whereinthe fusion protein is soluble.
 5. The pharmaceutical composition ofclaim 1, further comprising an adjuvant.
 6. A polynucleotide thatencodes a polypeptide comprising an amino acid sequence consistingessentially of amino acids 185-197, 209-221, 288-307 or 430-449 ofU_(L)48 (SEQ ID NO: 2), amino acids 14-22, 21-35, 45-59, 49-57, 49-63,105-190, 177-220 or 193-208 of U_(L)49 (SEQ ID NO: 3), or amino acids92-101, 92-105,288-307 or 743-751 of ICP0 (SEQ ID NO: 1).
 7. A vectorcomprising the polynucleotide of claim
 6. 8. A host cell transformedwith the vector of claim
 7. 9. A method of producing an HSV polypeptidecomprising culturing the host cell of claim 8 and recovering thepolypeptide so produced.
 10. An HSV polypeptide produced by the methodof claim
 9. 11. A pharmaceutical composition comprising thepolynucleotide of claim 6 and a pharmaceutically acceptable carrier. 12.The pharmaceutical composition of claim 11, further comprising anadjuvant.
 13. A pharmaceutical composition comprising a polynucleotidethat encodes a U_(L)48, U_(L)49 or ICP0 protein, and a pharmaceuticallyacceptable carrier.
 14. The pharmaceutical composition of claim 13,further comprising an adjuvant.
 15. A recombinant virus geneticallymodified to express an amino acid sequence consisting essentially ofamino acids 185-197, 209-221, 288-307 or 430-449 of U_(L)48 (SEQ ID NO:2), amino acids 14-22, 21-35, 45-59, 49-57, 49-63,105-190,177-220 or193-208 of U_(L)49 (SEQ ID NO: 3), or amino acids 92-101, 92-105,288-307 or 743-751 of ICP0 (SEQ ID NO: 1).
 16. The recombinant virus ofclaim 15 which is a vaccinia virus, canary pox virus or adenovirus. 17.A pharmaceutical composition comprising the virus of claim 15 and apharmaceutically acceptable carrier.
 18. The pharmaceutical compositionof claim 17, further comprising an adjuvant.
 19. A method of producingimmune cells directed against HSV comprising contacting an immune cellwith an antigen-presenting cell, wherein the antigen-presenting cell ismodified to present an epitope included in a U_(L)48, U_(L)49 or ICP0protein.
 20. The method of claim 19, wherein the epitope comprises aminoacids 185-197, 209-221, 288-307 or 430-449 of U_(L)48 (SEQ ID NO: 2),amino acids 14-22, 21-35, 45-59, 49-57, 49-63, 105-190, 177-220 or193-208 of UL49 (SEQ ID NO: 3), or amino acids 92-101, 92-105, 288-307or 743-751 of ICP0 (SEQ ID NO: 1).
 21. The method of claim 19, whereinthe immune cell is a T cell.
 22. The method of claim 21, wherein the Tcell is a CD4+ or CD8+ T cell.
 23. An immune cell produced by the methodof claim
 19. 24. A method of killing an HSV infected cell comprisingcontacting an HSV infected cell with the immune cell of claim
 23. 25. Amethod of inhibiting HSV replication comprising contacting a herpessimplex virus with the immune cell of claim
 23. 26. A method ofenhancing secretion of antiviral or immunomodulatory lymphokinescomprising contacting an HSV infected cell with the immune cell of claim23.
 27. A method of enhancing production of HSV-specific antibodycomprising contacting an HSV infected cell in a subject with the immunecell of claim
 23. 28. A method of enhancing proliferation ofHSV-specific T cells comprising contacting the HSV-specific T cells withan isolated polypeptide that comprises an epitope included in a U_(L)48,U_(L)49 or ICP0 protein.
 29. A method of inducing an immune response toan HSV infection in a subject comprising administering the compositionof claim 1 to the subject.
 30. A method of inducing an immune responseto an HSV infection in a subject comprising administering thecomposition of claim 11 to the subject.
 31. A method of treating orpreventing an HSV infection in a subject comprising administering thecomposition of claim 1 to the subject.
 32. A method of treating orpreventing an HSV infection in a subject comprising administering thecomposition of claim 11 to the subject.
 33. A method of treating orpreventing an HSV infection in a subject comprising administering anantigen-presenting cell modified to present an epitope included in aU_(L)48, U_(L)49 or ICP0 protein to the subject.